News from LabRulezLCMS Library - Week 15, 2025

<p><strong>The goal of this study was to develop and validate a novel LC-MS/MS assay capable of accurately measuring low concentrations of creatinine in sweat and saliva. These non-invasively obtainable biofluids are explored as alternatives to blood for continuous monitoring of kidney function, particularly during haemodialysis.</strong></p><p>By analyzing samples from forty dialysis patients, the study demonstrated strong correlations between creatinine levels in sweat, saliva, and plasma. The validated assay showed high accuracy and precision, with a low limit of quantitation. These findings support the potential use of sweat and saliva in wearable biosensor systems, offering a foundation for patient-friendly, non-invasive kidney monitoring technologies.</p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537"><strong>A novel LC-MS/MS assay for low concentrations of creatinine in sweat and saliva to validate biosensors for continuous monitoring of renal function</strong></a></h4><p>Sophie Adelaars, Chyara S.M. Lapré, Patricia Raaijmakers, Constantijn J.A.M. Konings, Massimo Mischi, R. Arthur Bouwman, Daan van de Kerkhof</p><p>Journal of Chromatography B, Volume 1252, 2025, 124444</p><p><a target="_blank" rel="noopener noreferrer" href="https://doi.org/10.1016/j.jchromb.2024.124444">https://doi.org/10.1016/j.jchromb.2024.124444</a></p><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><p>Accurately and timely monitoring of renal function is critical for managing patients at risk of acute kidney injury (AKI), a condition that affects approximately 12% of hospitalized patients <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0005"><span>[1]</span></a> and up to 57% of ICU patients <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0010"><span>[2]</span></a>. In about 20% of these cases, progression occurs towards higher stages of AKI, with significantly longer periods of hospitalization, increased mortality rates and more frequent need for invasive renal replacement therapy <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0005"><span>[1]</span></a>. This progression could potentially be delayed when patients are proactively treated based on frequent or continuous monitoring of renal function related biomarkers, such as creatinine.</p><p>As renal failure can progress rapidly, frequent venepuncture for creatinine analysis is required. Continuous monitoring of creatinine concentration can possibly improve effective patient management by further minimizing diagnostic delay. Recent developments in microfluidic devices with biosensors have sparked interest in alternative biofluids, such as sweat and saliva, as non-invasive matrices for continuous creatinine monitoring <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0015"><span>[3]</span></a>. The existing literature demonstrates moderate to high correlation coefficients between saliva and plasma creatinine concentrations, implying a potential utility of saliva in the diagnosis and monitoring of patients with AKI <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0020"><span>[4]</span></a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0025"><span>[5]</span></a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0030"><span>[6]</span></a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0035"><span>[7]</span></a>. However, the correlation between sweat and plasma creatinine concentrations remains poorly investigated. Contrarily to the relatively high urea concentrations in sweat, which supposedly contributes significantly to urea clearance in renal failure <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0040"><span>[8]</span></a>, <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0045"><span>[9]</span></a>, sweat creatinine concentration generally remains &lt;15&nbsp;µmol/L. Besides the fact that this complicates correlation studies, it also suggests that creatinine clearance via sweat is physiologically irrelevant in healthy conditions as well as in renal failure, as hypothesized by Al-Tamer et al. [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0080"><span>16</span></a>].</p><p>To enable accurate quantification of creatinine in these alternative biofluids, especially in sweat where concentrations are low, the development of a highly sensitive and specific analytical method is required. Conventional enzymatic assays, with limits of quantification (LoQ) around 10&nbsp;µmol/L, lack the sensitivity needed for this purpose. Our developed LC-MS/MS assay, with its lower limit of quantification, aims to serve as a reference method to validate emerging biosensors for real-time, continuous renal function monitoring. These biosensors hold the potential to minimize diagnostic delays, offering more immediate insight into renal function status [<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0050"><span>10</span></a>].</p><p>In this study, we aimed to develop and validate a novel LC-MS/MS assay that meets these sensitivity requirements with a significantly lower LoQ than conventional enzymatic methods. This work builds upon our previous research, in which we explored the concentrations of urea and creatinine in sweat and saliva from the same cohort of patients <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#b0055"><span>[11]</span></a>. However, the enzymatic assay used in that study faced limitations; it was not sensitive enough to quantify creatinine in many samples due to the assay’s LoQ, and it required larger sweat volumes than practically collected. The current study seeks to address these limitations by presenting an LC-MS/MS method that requires smaller sample volumes while offering a lower LoQ. Additionally, the assay can be utilized as a reference method to validate the performance of emerging biosensors aimed at continuous monitoring of renal function. Sweat and saliva samples were collected from forty haemodialysis patients to explore the feasibility of using sweat and saliva for continuous, non-invasive monitoring of renal function.</p><h4><strong>2. Methods</strong></h4><h5><strong>2.1.3. LC-MS/MS</strong></h5><p>The analysis was performed using an <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/191"><strong>Acquity Ultraperformance Liquid Chromatograph (UP-LC) </strong></a>from Waters (Milford, MA, USA). 5&nbsp;µL of sample was injected onto a 4&nbsp;mm&nbsp;×&nbsp;3&nbsp;mm strong cation exchange SecurityGuard column (Phenomenex, Macclesfield, UK). Every measurement started with 100% mobile phase A (water&nbsp;+&nbsp;2&nbsp;mmol/L ammoniumacetate&nbsp;+&nbsp;0.1% FA), stepping up at 0.40&nbsp;min to 100% mobile phase B (water&nbsp;+&nbsp;50&nbsp;mmol/L ammoniumacetate&nbsp;+&nbsp;10% MeOH + 0.1% FA), holding for 0.20&nbsp;min before returning to initial conditions until a total runtime of 1.10&nbsp;min, with a flow rate of 0.6&nbsp;mL/min and a column temperature of 30&nbsp;°C. A typical chromatograms obtained for sweat samples is shown in Supplemental Fig. S1.</p><p>An <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products?category=20&amp;manufacturerItems=5&amp;search=xevo+tq-s"><strong>Acquity Xevo TQ-S triple MS/MS</strong></a> from Waters (Milford, MA, USA) was used with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/635"><strong>MassLynx software</strong></a> from Waters (Milford, MA, USA) and results were analysed with <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?manufacturerItems=5&amp;search=TargetLynx+software"><strong>TargetLynx software</strong></a> from Waters (Milford, MA, USA). Multiple Reaction Monitoring (MRM) in the positive electrospray ionization (ESI+) mode was used as MS acquisition mode. MS settings were set as follows: capillary voltage: 2.40&nbsp;kV, cone voltage: 40&nbsp;V, desolvation temperature: 200&nbsp;°C, source temperature: 150&nbsp;°C, desolvation gas flow: 550&nbsp;L/h, nebulizer gas: 7.0&nbsp;bar, collision gas: 0.15&nbsp;mL/min, dwell time: 0.063&nbsp;s. The specific transitions and collision voltage were monitored and optimized in Multiple Reaction Monitoring (MRM) (Supplemental Table S1).</p><h4><strong>3. Results</strong></h4><h5><strong>3.4. Creatinine concentrations and correlation</strong></h5><p>The results of the creatinine concentrations in plasma, sweat and saliva are summarized in <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#t0010"><span>Table 2</span></a> and visualized in <a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#f0010"><span>Fig. 2</span></a>. Median creatinine concentrations in plasma decreased significantly during haemodialysis, from 677&nbsp;µmol/L to 232&nbsp;µmol/L (p&nbsp;&lt;&nbsp;0.001). Similarly, the concentrations decreased significantly in saliva, from 59.2&nbsp;µmol/L to 14.0&nbsp;µmol/L (p&nbsp;&lt;&nbsp;0.001), and in sweat, from 41.2&nbsp;µmol/L to 18.4&nbsp;µmol/L (p&nbsp;&lt;&nbsp;0.001).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Journal_of_Chromatography_B_Volume_1252_2025_124444_Fig_2_Creatinine_concentrations_at_the_start_and_at_the_end_of_the_haemodialysis_cycle_14f23fe87d.jpg" alt="Journal of Chromatography B, Volume 1252, 2025, 124444: Fig. 2. Creatinine concentrations at the start and at the end of the haemodialysis cycle, measured in plasma, sweat and saliva. All creatinine concentrations in sweat and saliva were significantly (***) lower than in plasma. *** = p < 0.001." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Journal_of_Chromatography_B_Volume_1252_2025_124444_Fig_2_Creatinine_concentrations_at_the_start_and_at_the_end_of_the_haemodialysis_cycle_14f23fe87d.jpg 245w," sizes="100vw" width="1600" height="895"></p><p>The mean sweat-to-plasma ratio was 0.071 (95% Confidence Interval (CI): 0.005–0.138). This ratio appeared to follow a decreasing pattern with increasing plasma creatinine concentrations (<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#f0015"><span>Fig. 3</span></a>A). The mean saliva-to-plasma ratio was 0.109 (95% CI: −0.066 to 0.283) and appeared constant across the entire range of measured plasma creatinine concentrations (<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#f0015"><span>Fig. 3</span></a>B).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Journal_of_Chromatography_B_Volume_1252_2025_124444_Fig_3_A_The_mean_sweat_to_plasma_was_0_071_and_appeared_to_follow_a_decreasing_pattern_with_increasing_plasma_creatinine_concentrations_75af5c371a.jpg" alt="Journal of Chromatography B, Volume 1252, 2025, 124444: Fig. 3. (A) The mean sweat-to-plasma was 0.071 and appeared to follow a decreasing pattern with increasing plasma creatinine concentrations. (B) The mean saliva-to-plasma ratio was 0.109 and appears constant across the range of the measured plasma creatinine concentration." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Journal_of_Chromatography_B_Volume_1252_2025_124444_Fig_3_A_The_mean_sweat_to_plasma_was_0_071_and_appeared_to_follow_a_decreasing_pattern_with_increasing_plasma_creatinine_concentrations_75af5c371a.jpg 245w," sizes="100vw" width="1600" height="787"></p><p>The Spearman’s correlation coefficient between creatinine concentrations in plasma with sweat was 0.68 (95% confidence interval (CI): 0.49–0.81, p&nbsp;&lt;&nbsp;0.001), while the correlation between creatinine concentrations in plasma with saliva was 0.80 (95% CI: 0.71–0.85, p&nbsp;&lt;&nbsp;0.001) (Supplemental Fig. S6). The results of the Passing–Bablok regression analysis, assessing the agreement between plasma and sweat and saliva creatinine concentrations, are also visualized in Supplemental Fig. S6.</p><h5><strong>3.5. Sweat rate dependency</strong></h5><p>Multivariable linear regression analysis to predict the plasma creatinine concentration based on sweat creatinine concentration revealed a significant positive effect of collected sweat volume (p&nbsp;&lt;&nbsp;0.001) (<a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S1570023224004537#f0020"><span>Fig. 4</span></a>). To enable visualization, sweat volumes were fixed at specific values (2, 5, 6, and 10&nbsp;µmol/L), and corresponding regression lines were plotted as examples. Specifically, higher sweat volumes were associated with lower concentrations in sweat and higher predicted concentrations in plasma. However, there was no direct correlation between the concentrations in sweat and sweat volume (<i>r</i>: −0.26, p&nbsp;&lt;&nbsp;0.05, R<sup>2</sup><sub>adj</sub>: 0.06); therefore, the concentrations in sweat were not corrected for the collected volume (Supplemental Fig. S7).</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/Journal_of_Chromatography_B_Volume_1252_2025_124444_Fig_4_Multivariable_linear_regression_analysis_b37b0335e5.jpg" alt="Journal of Chromatography B, Volume 1252, 2025, 124444: Fig. 4. Multivariable linear regression analysis revealed a significant effect of the collected sweat volume on the prediction of plasma creatinine concentration with sweat creatinine concentration. Regression lines at different sweat volumes are visualized in blue. Sweat volumes of 10, 40 and 70 µL were filled in as constants in the multivariable linear regression formula (y = ax1 + bx2 + c, where y = plasma creatinine concentrations, x1 = sweat creatinine concentration, x2 = sweat volume and a, b and c are constants determined by the model). To demonstrate, a sweat creatinine concentration of 40 µmol/L without taking volume into account would predict a plasma creatinine concentration of ±633 µmol/L (black square). With the volumes 10 µL, 40 µL and 70 µL, the plasma concentration would be predicted to be 528, 700 and 872 µmol/L (red squares), respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)" srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_Journal_of_Chromatography_B_Volume_1252_2025_124444_Fig_4_Multivariable_linear_regression_analysis_b37b0335e5.jpg 132w," sizes="100vw" width="1378" height="1627"></p><h4><strong>4. Discussion</strong></h4><p>In summary, this study builds upon our previous research by employing a novel LC-MS/MS assay to quantify creatinine concentrations in sweat and saliva, enabling the measurement of lower analyte concentrations than conventional enzymatic methods. While our earlier work was the first to explore urea and creatinine in these biofluids within this patient cohort, this follow-up study provides a more comprehensive dataset that enhances our understanding of the correlations between these non-invasive biomarkers and plasma creatinine. The strong correlations observed support the potential of sweat and salivary creatinine as valid indicators of renal function. Moreover, these findings might pave the way for future applications in validating wearable biosensors for continuous monitoring of patients with AKI in low-resource settings, ultimately improving patient care through real-time assessment and timely intervention.</p>

A novel LC-MS/MS assay for low concentrations of creatinine in sweat and saliva to validate biosensors for continuous monitoring of renal function

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 22nd September 2025? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you application notes by <strong>Agilent Technologies, Shimadzu </strong>and <strong>Waters Corporation </strong>and<strong> </strong>poster by <strong>Thermo Fisher Scientific / HPLC Symposium!</strong></p><h3><strong>1. Agilent Technologies: Quantitation of N-Nitroso Nebivolol in Nebivolol Tablet Formulation</strong></h3><p><strong>Using an </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D+triple+quadrupole"><strong>Agilent 6495D triple quadrupole LC/MS system</strong></a></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_nebivolol_tablet_formulation_6495_qqq_lc_ms_system_5994_8053en_agilent_e7f21eb12f.pdf"><strong>Full PDF for download</strong></a></li></ul><p>N-Nitroso Nebivolol is a NDSRI of Nebivolol, a beta-1 adrenergic receptor antagonist commonly used in the treatment of hypertension and heart failure. In the carcinogenic potency categorization published by the FDA, N-Nitroso Nebivolol has been categorized as Class 4, indicating the maximum allowable intake limit for the impurity to be 1,500 ng/day. Nebivolol has maximum daily dosage limit of 40&nbsp;mg for adults.</p><p>Considering a maximum daily dose of 40&nbsp;mg and allowable intake of 1,500&nbsp;ng/day for the nitrosamine impurity in the formulation under study, the specification limit of N-Nitroso Nebivolol has been calculated as 22.5 ng/mL for the 0.6 mg/mL test concentration used during this study.&nbsp;</p><p>This application note demonstrates a highly specific, sensitive, and reproducible method for the quantitation of N-Nitroso Nebivolol in a 20 mg Nebivolol tablet formulation. The method uses a 1290 Infinity II LC coupled to a 6495D triple quadrupole LC/MS/MS. The multiple reaction monitoring (MRM) conditions and ion source parameters were automatically optimized using the optimizer tool available with MassHunter Acquisition software to maximize the sensitivity of the target analyte. The API peaks were diverted to waste with the help of an integrated, software-controlled diverter valve during quantitation of the&nbsp;impurity.</p><h4><strong>Experimental</strong></h4><h5><strong>Equipment&nbsp;</strong></h5><p>The analysis was performed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+II+LC"><strong>1290 Infinity II LC system</strong></a> coupled to a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6495D+triple+quadrupole"><strong>6495D triple quadrupole LC/MS/ MS</strong></a>. The LC system was equipped with the following&nbsp;modules: – Agilent 1290 Infinity II high-speed pump (G7120A) – Agilent 1290 Infinity II multisampler (G7167B) – Agilent 1290 Infinity II multicolumn thermostat (G7116B) – Agilent 1290 Infinity II diode array detector (G7117A)</p><h4><strong>Conclusion&nbsp;</strong></h4><p>A highly sensitive and robust MRM method was developed for the quantitation of N-Nitroso Nebivolol in 20&nbsp;mg Nebivolol tablet formulation using an Agilent 6495D triple quadrupole LC/MS system. The chromatographic method developed provided good separation between the analyte and the API. The method showed LOD and LOQ of 2 and 5 pg/mL, respectively. The minimum signal-to-noise ratio at the LOQ level was found to be more than 50:1 (Peak to Peak). The calibration curve from 5 pg/mL to 50&nbsp;ng/mL was found to be linear, with 1/x weighting. R and R2 values were 0.999 and 0.997, respectively. Spike recovery analysis showed the efficiency of sample extraction with a recovery percentage of 116.8% in API, 108.3% in placebo, and 111.8% in tablet formulation at the spiking level of 200 pg/mL. The method was found to be highly reproducible at the LOQ level, while the %RSD value for the area response of seven replicate injections was approximately 3.1%.</p><h3><strong>2. Shimadzu: Efficient Method Development for Synthetic Peptide and Related Impurities</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_01_00780_en_4d0aa014ea.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>User Benefits</strong></h5><ul><li>LabSolutions MD improves the efficiency of the entire workflow for method development of synthetic peptide and impurities.&nbsp;</li><li>Screening for multiple mobile phases and columns can be automated using mobile phase and column switching valves.&nbsp;</li><li>Molecular weights of peptides and related impurities can be estimated and accurately tracked with LCMS-2050, a single quadrupole mass spectrometer.</li></ul><p>Peptide therapeutics, characterized by specific amino acid sequences crucial to their function, can be synthesized chemically, similar to small molecule drugs. The production of synthetic peptides involves multiple steps, including deprotection, activation, coupling, and the cleavage of the final sequence from the solid support. Impurities, such as those resulting from premature chain termination or missing amino acids, can affect the safety and efficacy of the final product. Therefore, these impurities must be separated by liquid chromatography (LC). In LC analysis, selecting the appropriate mobile phase and column across a wide range of combinations is critical for achieving optimal separation, as it significantly impacts the separation. However, since the separation pattern varies depending on the peptide chain lengths, amino acid compositions, and presence of modifications, optimizing separation for each peptide sequence is time-intensive. This study describes how to efficiently find the best separation conditions for peptides and related impurities utilizing LabSolutions MD, a dedicated software for supporting method development, through both screening and optimization phases.</p><h4><strong>Experimental</strong></h4><ul><li><strong>LC System: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera+X3"><strong>Nexera X3</strong></a> (Method Scouting System)</li><li><strong>MS System: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-2050"><strong>LCMS-2050</strong></a></li></ul><h4><strong>Automated Peak Tracking by LCMS-2050&nbsp;</strong></h4><p>LC chromatograms obtained with a gradient time of 5 min, flow rate of 0.6 mL/min, and initial concentration of 5 % and 15 %, along with molecular weights for FLP and impurities, are shown in Fig. 18. The UV spectra for each impurity are displayed in Fig. 19. The similarity between UV spectra of Met(O2), p.A1del, p.A1_E2del, p.A1_K3del, and p.A1_D5del is greater than 0.99, suggesting that peak tracking based on UV spectra would be challenging. In contrast, LabSolutions MD enables peak tracking based on molecular weights acquired by LCMS-2050, facilitating accurate identification of compounds with similar UV spectra (Fig. 18). The estimated molecular weights for each compound show small mass errors compared to the theoretical values (Table 3), which can be used to confirm the molecular weights of known compounds as well as to approximate the molecular weights of unknown impurities.</p><h4><strong>Conclusion</strong></h4><p>The separation patterns of synthetic peptides vary depending on mobile phase composition, column type, and various LC parameters, such as gradient conditions, column oven temperature, and flow rate. Separation behavior can also differ based on peptide structure, including length, amino acid composition, and the presence of modifications. Therefore, it is necessary to optimize the separation for each peptide sequence individually. However, optimizing analytical conditions through numerous analyses and data processing can be time-consuming. LabSolutions MD automates the entire workflow, including generating analysis schedules, preparing mobile phases, and processing the data, thanks to functionalities such as automated peak tracking, ranking of chromatograms, and design space visualization. This article introduced a case where the optimal separation conditions for synthetic peptides were efficiently identified through screening and optimization phases. LabSolutions MD also offers fully automated gradient optimization by AI algorithm to meet user-defined criteria. For more details, please refer to the application news, “Automatic Optimization of Gradient Conditions by AI Algorithm on Synthetic Peptide and Impurities: 01-00814”.</p><h3><strong>3. Thermo Fisher Scientific / HPLC Symposium: Sensitive cationic lipids impurities analysis with quantitation by charged aerosol detection and simultaneous mass confirmation by MS</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_sensitive_cationic_lipids_impurities_analysis_quantitation_po_en_3dacf61554.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Cationic lipid is a critical component in lipid nanoparticle formulations for delivering nucleic acids such as mRNA, siRNA, antisense oligonucleotides etc. [2] Cationic lipids are amphiphilic molecules that possess a hydrophilic region, a hydrophobic region, and a linker structure connecting the two. [1] Charged aerosol detector (CAD) and evaporative light scattering detectors (ELSD) are preferred for characterizing lipid nanoparticle components, with CAD being the better choice for impurity quantification due to its higher sensitivity [3]. In a previous study, an inverse gradient method was developed to quantify impurities in cationic lipids raw materials.[1] To demonstrate the inverse gradient method on CAD HP, the same cationic lipids -- R-DOTAP, DLin-KC2- DMA and ALC-0315 -- were measured to this experiment as in Application note 003384 [1]. New diverter valve and software features were showcased, along with method’s sensitivity and repeatability.</p><h4><strong>Materials and methods</strong></h4><p>Using the inverse gradient method from Application note 003384 [1], to demonstrate impurity analysis in cationic lipids on the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2176"><strong>Thermo Scientific Vanquish Charged Aerosol Detector HP (CAD HP)</strong></a> integrated in the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/743"><strong>Thermo Scientific Vanquish Flex Inverse Gradient UHPLC System</strong></a>, with parallel mass confirmation by the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/413"><strong>Thermo Scientific ISQ EM Single Quadrupole Mass Spectrometer</strong></a>.&nbsp;</p><p><strong>Methods:</strong> The workflow uses a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/743"><strong>Vanquish Flex Inverse Gradient LC system</strong></a> with Vanquish CAD HP and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1970"><strong>Thermo Scientific Hypersil GOLD C8 column</strong></a>.</p><h5><strong>Data Analysis&nbsp;</strong></h5><p><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/76"><strong>Thermo Scientific Chromeleon Chromatography Data System (CDS)</strong></a> 7.2.10 was used for data acquisition and analysis.</p><h4><strong>Conclusions&nbsp;</strong></h4><p>The CAD HP integrates perfectly into the Vanquish Inverse Gradient LC system and offers high sensitivity and excellent repeatability for quantitative cationic lipid impurity analysis, while the ISQ EM MS provides straightforward mass confirmation with high repeatability, all under compliance-ready conditions.</p><h3><strong>4. Waters Corporation: Quantitative Analysis of Four Long-Chain Lysophosphatidylcholines (LPCs) in Dried Blood Spot using Liquid Chromatography Tandem Mass Spectrometry for Clinical Research</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720008826_en_dc154872b6.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits</strong></h5><ul><li>Good chromatographic resolution of all forms</li><li>Short extraction time compared to traditional methods for lipid extraction</li><li>Short analysis injection-to-injection 7 minutes</li></ul><p>Historically, very long chain fatty acids have been quantified using direct transesterification and gas chromatography methods which involve time-consuming and labor-intensive sample preparation and analysis. Consequently, simpler LC-MS/MS methods have been reported for the measurement of long-chain LPCs (LPCs generic chemical structure, Figure 1) in DBS. Interferences have been reported using positive ESI, thus researchers have developed methodologies utilizing negative ESI to circumvent isobaric interferences when there is lack of chromatographic resolution. Here a UHPLC-MS/MS method is described using acetate adducts for the reliable analysis of four long-chain LPCs in DBS for clinical research.&nbsp;</p><p>A UHPLC-MS/MS method for quantifying a panel of LPCs (C20:0, C22:0, C24:0, and C26:0-LPCs) was developed. The mobile phase composition consisted of a mixture of water, acetonitrile, and isopropanol with 5 mM ammonium acetate as a modifier. The DBS sample extraction used a working solution prepared with the deuterated LPC internal standards in methanol. The method was developed with an ACQUITY UPLC I-Class PLUS System (Fixed Loop) using an ACQUITY Premier CSH C18 Column, which provided baseline resolution of endogenous isobaric interferences for the saturated forms of the targeted LPCs. The detector was a Xevo TQ-S micro Mass Spectrometer operated in negative ESI mode. This methodology resulted in a total analysis time (injection-to-injection) of 7 minutes.</p><h5><strong>LC Conditions&nbsp;</strong></h5><ul><li><strong>LC system: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/199"><strong>ACQUITY UPLC I-Class PLUS FL&nbsp;</strong></a></li><li><strong>System Detection: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/209"><strong>Xevo TQ-S micro Mass Spectrometer&nbsp;</strong></a></li><li><strong>Plate:</strong> Waters 96-well Sample Collection Plate, 350 µL&nbsp;</li><li><strong>Column(s): </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1952"><strong>ACQUITY Premier CSH C18 Column</strong></a>, 2.1 mm x 50 mm, 1.7 µm (p/n: 186009460)</li></ul><h5><strong>MS Conditions&nbsp;</strong></h5><ul><li><strong>MS system:</strong> Xevo TQ-S micro Mass Spectrometer</li></ul><h5><strong>Data Management&nbsp;</strong></h5><ul><li><strong>MS software: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/635"><strong>MassLynx™ v4.2 Software SCN 1045&nbsp;</strong></a></li><li><strong>Informatics: </strong>TargetLynx™ XS v4.2 Software SCN1045 and QUAN Review</li></ul><h4><strong>Results and Discussion&nbsp;</strong></h4><p>During method development, different types of column chemistry, mobile phase composition, and ESI polarity modes were assessed to optimize chromatographic peak shape, analytical sensitivity and manage interferences. In positive mode, ammonium formate favored the formation of m/z 184 and m/z 104 as daughter ions for all the LPCs. The m/z 184 ion lacks specificity because most phospholipids share this moiety, so the ion m/z 104 was selected as quantifier. Having evaluated conditions in positive mode for the analysis of the four saturated LPCs, it was observed that some existing interferences were not only affecting the parent masses of the analytes but their corresponding internal standard parent ion masses too. Hence, negative ionization using ammonium acetate as modifier was assessed for the detection of LPCs. Acetate adducts [M+CH3COO]- were the most abundant parent ion masses, generated under these conditions for each LPC. The acyl-chain product ions generated in negative ionization reduce the detection of interferences coming from other phosphocholine headgroups. The latter detection conditions were used for the validation of the method presented herein.</p><h4><strong>Conclusion&nbsp;</strong></h4><p>An LC-MS/MS clinical research method for measuring C20:0, C22:0, C24:0, and C26:0 LPCs with the ACQUITY UPLC I-Class PLUS FL System, and the Xevo TQ-S micro Mass Spectrometer that:</p><ul><li>Demonstrates good linearity, with no significant carryover, or matrix effects</li><li>Offers good precision with a total reproducibility and repeatability <span>≤</span>10% CV</li><li>Offers reliability in the identification of LPCs from interfering isobaric compounds Provides good selectivity and analytical sensitivity in negative mode and is relatively fast as approximately 220 samples can be analyzed in 24 hours</li></ul>

News from LabRulezLCMS Library - Week 39, 2025

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 19th January 2026? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you application notes by <strong>Agilent Technologies, Shimadzu, Waters Corporation </strong>and poster by<strong> Thermo Fisher Scientific!</strong></p><h3><strong>1. Agilent Technologies: Automated Bioprocess Monitoring with Agilent 1290 Infinity III Bio Online LC Solutions</strong></h3><p><strong>Process analytics of L-alanine production using genetically modified </strong><i><strong>Vibrio natriegens</strong></i></p><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_automated_bioprocess_monitoring_1290_infinity_bioonline_solutions_5994_8865en_agilent_9f33de6ddf.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Bioprocess engineering is a challenging field because of the constantly changing nature of bioprocess models which make them difficult to validate.1 Consequently, process control needs to be developed at various levels using strategies tailored to these specific requirements.1 The performance and effectiveness of the bioreactor are determined by key performance indicators such as product yield, titer, production rate, and quality of the product.1 These factors can be quickly influenced by changes in process variables and the raw materials used.1,2 The ability to react quickly to problems is therefore crucial for preserving ongoing production, a factor that can be particularly significant in bioprocesses due to the substantial cost and time investmented.1,2 Such challenges can be tackled by constantly monitoring the bioprocess, which requires the ability to analyze and collect data quickly. Therefore, monitoring is of the utmost importance.&nbsp;</p><p>This application note describes the advantages of using an Agilent 1290 Infinity III Bio Online LC solution with Agilent Online LC Monitoring software. A bioreactor was interfaced with a Flownamics Seg-Flow S3 sampling system for aseptic withdrawal and an Agilent 1290 Infinity III Bio Online LC to make an hourly analysis.&nbsp;</p><p>This is a fully automated approach for taking sterile samples from a bioreactor and preparing or diluting them. In addition, the 1290 Infinity III Bio Online LC solution displays data in near real time, which is a crucial element for effective bioprocess&nbsp;monitoring.</p><h4><strong>Experimental&nbsp;</strong></h4><h5><strong>Equipment&nbsp;</strong></h5><p>The <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=1290+Infinity+III+Bio"><strong>1290 Infinity III Bio </strong></a>Online LC comprised the following&nbsp;modules:&nbsp;</p><ul><li>Agilent 1290 Infinity III Bio Flexible Pump (G7131A) with an InfinityLab Quick Change Bio inline filter assembly (part&nbsp;number 5067-1607), 2.1 mm id, 0.2 µm pore size&nbsp;</li><li>Agilent 1290 Infinity III Bio Online Sample Manager (G3167B) with Sample Thermostat (option #101) clustered with Agilent 1290 Infinity Valve Drive (G1170A), featuring a reactor valve pod (part number 5067-6680) and Online LC Monitoring Software&nbsp;</li><li>Agilent 1290 Infinity III Multicolumn Thermostat (G7116B) equipped with an Agilent Quick Connect Bio Heat Exchanger Std. (G7116-60071)&nbsp;</li><li>Agilent 1290 Infinity III Diode Array Detector (DAD) (G7117B), equipped with a Bio Max-Light Cartridge Cell (G4212-6008), 10 mm, 1 µL&nbsp;</li><li>Agilent 1260 Infinity High-Performance Degasser (G4225A) is connected between S1 (transport solvent) solvent line and Agilent 1290 Infinity III Bio Online Sample Manager (G3167B)&nbsp;</li></ul><h5><strong>Software&nbsp;</strong></h5><ul><li>Agilent <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=OpenLab+CDS"><strong>OpenLab CDS</strong></a> version 2.8 or later versions&nbsp;</li><li>Agilent Online LC Monitoring Software version 1.3 or later&nbsp;versions&nbsp;</li><li>Flownamics FlowWeb control&nbsp;software</li></ul><h5><strong>Instrumental configuration&nbsp;</strong></h5><p>The 1290 Infinity III Bio Online LC solution was used with a Seg-Flow interface for automatic sampling and online analysis (see Figure 1). The Seg-Flow interface was used to withdraw samples from a bioreactor through a Flownamics F-Series 310 FISP probe with a ceramic membrane filter (0.2&nbsp;µm pore size) from a 5 L bioreactor. The drawn samples were sent through the built-in sample collection cup on the Seg‑Flow to the LC and the samples were retained into vials. The collected samples were then automatically derivatized by using an injector program and subsequently analyzed. A more detailed description of the instrumental configuration has been published.<sup>3</sup></p><h4><strong>Conclusion&nbsp;</strong></h4><p>For online bioprocess monitoring, a fully automated setup consisting of a bioreactor, a Flownamics sampling system, and an Agilent 1290 Infinity III Bio Online LC solution was successfully established. This setup is capable of taking aseptic samples from a bioreactor, preparing, analyzing, and processing samples without the need for manual interaction. In addition, the added value of the Agilent Online LC Monitoring software was evident in the display method and the ability to track it quickly.</p><h3><strong>2. Shimadzu: Analysis of Oligonucleotide Impurities Using Single Quadrupole Mass Spectrometer</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/an_01_00974_en_c50bb67c7a.pdf"><strong>Full PDF for download</strong></a></li></ul><h4><strong>User Benefits</strong></h4><ul><li>Combination use of LCMS-2050 single quadrupole mass spectrometer and LabSolutions Insight Biologics provides comprehensive characterization of oligonucleotides and related impurities.</li><li>Purity calculations for target components can be performed even on poorly separated peaks by utilizing MS spectra simultaneously acquired with UV chromatogram.</li></ul><p>In recent years, oligonucleotide therapeutics have been paid attention as a novel drug discovery modality because of their rapid progress of development. During the synthesis of oligonucleotide therapeutics, impurities that have differing base chain lengths are generated along with the target oligonucleotide. To ensure the safety and efficacy of the drug, comprehensive detection and identification of these impurities are required.&nbsp;</p><p>Reversed phase ion-pair chromatography is often employed as a method to separate the target compound from co-existing impurities. However, it is often difficult to establish analytical conditions that provide compete separation of all impurities that are formed in synthesis process due to their similar structures and properties to the principal compound of active ingredient. Against this drawback, liquid chromatography-mass spectrometry (LC-MS) is commonly used for impurity detection and identification. For analysis of oligonucleotide impurities, quadrupole time-of-flight mass spectrometers are mainly used because of their high mass resolution and ability to perform sequence and structure estimations via MS/MS analysis. However, in quality control process, the use of a single quadrupole mass spectrometer is expected to be advantageous because of providing mass information with easy operation when impurity information is already known.&nbsp;</p><p>This article presents a simulated impurity analysis of synthetic oligonucleotide using the LCMS-2050 high-performance liquid chromatography-mass spectrometer and LabSolutions Insight Biologics.</p><h4><strong>Analytical conditions</strong></h4><p>The employed instrument setup was <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera+XS"><strong>Nexera XS inert</strong></a> and <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=LCMS-2050"><strong>LCMS-2050</strong></a>. HPLC conditions are shown in Table 2, and MS conditions are shown in Table 3. The conditions were set where n-1(5') co-eluted with FLP, while n-3(5') was separated (Fig. 1) to demonstrate impurity analysis performance.</p><h4><strong>Conclusion</strong></h4><p>The simulated sample consisting of mainly FLP and small amounts of n-1(5')/n-3(5') was analyzed using the LCMS-2050 and LabSolutions Insight Biologics software and obtained data were processed appropriately. Detection, identification, and quantitation at low concentrations were accomplished not only for n-3(5') well-separated from FLP but also for n-1(5') co-eluted with FLP. In case that establishing analytical conditions is difficult, Using this instrument setup and software will provide efficient quality confirmation of oligonucleotide therapeutics.</p><h3><strong>3. Thermo Fisher Scientific: Simultaneous Quantitation and Untargeted Discovery (SQUAD) workflow using multiple fragmentation techniques for quantitation and characterization of trace level nitrosamine in biological matrices</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_squad_workflow_multiple_fragmentation_techniques_po_en_94a5b27b12.pdf"><strong>Full PDF for download</strong></a></li></ul><p>N-Nitrosamines are a class of carcinogenic chemical compounds that pose threat to humans exogenously through matrices like drinking water, food, cosmetics, and endogenously by nitrosation in gastrointestinal tract, nitrate to nitrite conversion, etc. An accurate, reliable analytical method is critical for detection and quantification of these trace-level, polar and low molecular weight compounds to understand the association between their exposure and related diseases.&nbsp;</p><p>An optimized SQUAD workflow is presented that discovers unknown compounds in the matrix by using high resolution orbitrap and a sensitive linear ion trap detector that quantifies the low intensity nitrosamines in a single run. The workflow uses multiple fragmentation types – HCD, CID and UVPD and MSn to help characterize isomeric structures.</p><h4><strong>Methods</strong></h4><p>Serial dilution of Nitrosamine standards were subjected to Simultaneous Quantitation and Discovery (SQUAD) analysis using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/744"><strong>Thermo Scientific™ Vanquish™ Horizon UHPLC system</strong></a> and the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/561"><strong>Thermo Scientific™ Orbitrap IQ-X™ Tribrid™ mass spectrometer</strong></a> . Nitrosamine spiked matrices of human pooled urine and human Serum were subjected to SQUAD analysis.</p><h4><strong>Data Analysis&nbsp;</strong></h4><p>Spectra were evaluated for nitrosamines using Thermo Scientific™ FreeStyle™ software and annotated using Thermo Scientific™ <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1757"><strong>Mass Frontier™ 8.0 software</strong></a>. Untargeted data analysis was performed using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1759"><strong>Compound Discoverer 3.4 software</strong></a>. Calibration curves were generated using Thermo Scientific™ <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/77"><strong>TraceFinder™ 5.2 software</strong></a>.</p><h4><strong>Conclusions&nbsp;</strong></h4><ul><li>Detection limits as low ppb levels ( 0.2 ppb –0.5 ppb) and quantitation limits (0.2 ppb- 5 ppb ) in presence of the matrix were achieved for all seven nitrosamines by applying parallel ion trap analysis&nbsp;</li><li>SQUAD analysis was run on Orbitrap IQ-X Tribrid MS and achieved low ppb level quantitation in ion trap and identified unknown compounds through discovery analysis in Orbitrap.&nbsp;</li><li>Employing nano LC helped achieve lower limit of quantitation (5 ppt)</li></ul><h3><strong>4. Waters Corporation: Validated LC-MS/MS Method for Vitamin E Acetate in Cannabis Plants and Derived Products</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009082_en_c7a6fc6190.pdf"><strong>Full PDF for download</strong></a></li></ul><h5><strong>Benefits&nbsp;</strong></h5><ul><li>The validated LC-MS/MS method provides high sensitivity and specificity for VEA quantification in complex cannabis matrices, including flower and concentrates.&nbsp;</li><li>Minimal sample preparation reduces handling time and improves laboratory throughput, supporting efficient routine analysis.&nbsp;</li><li>The method demonstrates robust performance across multiple matrices, ensuring reliable detection even in lipid-rich, chemically diverse samples.</li><li>Use of Waters ACQUITY UPLC H-Class System and Xevo TQ-S micro Mass Spectrometer enables accurate quantitation well below regulatory requirements.&nbsp;</li><li>The validated method complies with Missouri DCR and DHSS regulatory limits, enabling laboratories to confidently monitor VEA in cannabis products.</li></ul><p>VEA is used as a diluent and viscosity modifier in certain cannabis-derived products, including vaping formulations. Following increased public health awareness during 2019, VEA became a marker of interest for quality control and regulatory surveillance across cannabis supply chains due to its potential impact on lung health. In addition to concentrates and vape liquids, trace residues may be present in infused products and processing intermediates. Therefore, reliable quantification of VEA in these chemically diverse, lipid-rich matrices requires an analytical method that is sensitive, selective, and robust against matrix effects. In February 2024, Missouri’s DCR revised the tolerance limit for VEA in cannabis products from 0.2 ppm to 5 ppm.<sup>1&nbsp;</sup></p><p>Liquid chromatography coupled with tandem quadrupole mass spectrometry (LC-MS/MS) provides the sensitivity and specificity required to distinguish VEA from endogenous cannabinoids, terpenes, and other lipophilic excipients, while also offering the sensitivity to support low-ppm and sub-ppm action limits. Unlike UVbased methods, where co-eluting chromophores may interfere, LC-MS/MS enables targeted multiple reaction monitoring (MRM) for confident identification and accurate quantitation in complex cannabis matrices.&nbsp;</p><p>This application note describes the validation of an LC-MS/MS method for the quantitation of VEA in two inhalable products, cannabis flower and cannabis concentrates. The method was developed and validated using the Waters ACQUITY UPLC H-Class System coupled with a Xevo TQ-S micro Triple Quadrupole Mass Spectrometer. Chromatographic separation was achieved with a reversed-phase ACQUITY UPLC BEH C18 Column. A streamlined sample preparation approach was applied for cannabis flowers and concentrates, which minimizes handling time and increases sample throughput.&nbsp;</p><p>The validation study was conducted at GPA. Method performance was evaluated for calibration range, linearity, LOD, LOQ, accuracy, precision, recovery, repeatability, and robustness. The results of these are evaluated against acceptance criteria set by State of Missouri DHSS and presented in this application note.</p><h4><strong>Experimental&nbsp;</strong></h4><ul><li><strong>LC system: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/198"><strong>ACQUITY UPLC H-Class Plus System&nbsp;</strong></a></li><li><strong>MS detection: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/209"><strong>Xevo TQ-S micro Mass Spectrometer&nbsp;</strong></a></li><li><strong>Column: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1952"><strong>ACQUITY UPLC BEH C18 Column</strong></a>, 2.1 x 50 mm, 1.7 µm (p/n: 186002350)</li><li><strong>Software:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/635"><strong>MassLynx™ 4.1 Software&nbsp;</strong></a></li><li><strong>Data processing: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=5&amp;search=TargetLynx"><strong>TargetLynx™</strong></a> Application</li></ul><h4><strong>Conclusion&nbsp;</strong></h4><p>The LC-MS/MS method developed and validated for VEA quantification in cannabis flower and concentrates demonstrates excellent linearity, accuracy, precision, and robustness, and meets the regulatory requirements. Calibration curves achieved R² values greater than 0.999, with LOD and LOQ well below the Missouri action limit of 5 ppm. Matrix-spiked recoveries across flower and concentrate samples were within the 70–130% acceptance range, and QC samples met all performance criteria, confirming method reliability. Robustness testing across analysts and matrices further validated the method’s reproducibility. Overall, this approach provides a streamlined, sensitive, and regulatory-compliant workflow for laboratories, enabling accurate monitoring of VEA to support quality control, consumer safety, and regulatory compliance in cannabis-derived products</p>

News from LabRulezLCMS Library - Week 04, 2026

Elucidating Unknown Organofluorine in Municipal Wastewater: A Mass Balance Approach including Fluorinated Pharmaceuticals

News from LabRulezLCMS Library - Week 27, 2026

<p><strong>Our Library never stops expanding. What are the most recent contributions to LabRulezLCMS Library in the week of 17th August 2026? Check out new documents from the field of liquid phase, especially HPLC and LC/MS techniques!</strong></p><blockquote><p><span style="font-size:20px;">👉 </span><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library"><span style="font-size:20px;"><strong>SEARCH THE LARGEST REPOSITORY OF DOCUMENTS ABOUT LCMS AND RELATED TECHNIQUES</strong></span></a></p></blockquote><blockquote><p><strong>👉 Need info about different analytical techniques?</strong> Peek into <a target="_blank" rel="noopener noreferrer" href="https://gcms.labrulez.com/library"><strong>LabRulezGCMS</strong></a> or <a target="_blank" rel="noopener noreferrer" href="https://icpms.labrulez.com/library"><strong>LabRulezICPMS</strong></a> libraries.</p></blockquote><p>This week we bring you posters by <strong>Agilent Technologies / ASMS, Thermo Fisher Scientific / ASMS </strong>and<strong> Shimadzu / ASMS </strong>and application note by<strong> Waters Corporation!</strong></p><h3><strong>1. Agilent Technologies / ASMS: Quantitation of N-Nitroso Duloxetine N-Nitrosamine Drug Substance Related Impurities (NDSRI) in API and Formulation by 6475 LC/TQ</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_drug_impurities_lc_tq_tp_402_asms_2026_en_agilent_b933b9bddf.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Nitrosamine impurities are nitroso derivatives where the nitroso group is attached to an amine. The active pharmaceutical ingredient (API) derived from a class of nitrosamines are known as N-nitrosamine drugsubstance-related impurities (NDSRI). Nitrosamine impurities in APIs and drug products pose safety concerns, even in small quantities, and thus are a major concern for drug makers [1].&nbsp;</p><p>Analytical methods must be sufficiently sensitive to quantify the nitrosamine of interest at levels as low as 10% of the specification limit and, where feasible, down to ≤5% of the specification limit, for the determination of the limit of detection. LC/MS/MS is an inherently selective and highly sensitive analytical technique that is well suited for the identification and quantification of trace-level impurities at extremely low concentrations. Consequently, LC/MS/MS has been widely adopted within the pharmaceutical industry, particularly for nitrosamine analysis [2-3].&nbsp;</p><p>Therefore, manufacturers must develop and qualify a dedicated analytical method that is appropriate for the specific sample matrix and targeted NDSRIs. An LC/MS/MS method based on multiple reaction monitoring (MRM) was developed for the detection and quantification of the N-nitroso duloxetine NDSRI impurity over a concentration range of 0.5 to 40 ppb (absolute standard concentration), using an atmospheric pressure chemical ionization (APCI) source on an Agilent 6475A LC/TQ instrument.</p><h4><strong>Experimental</strong></h4><ul><li><strong>MS:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=6475"><strong>6475 Triple Quadrupole LC/MS System</strong></a></li><li><strong>Column:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=10&amp;search=Poroshell+HPH+C18"><strong>Poroshell HPH C18</strong></a> (4.6*150mm, 2.7µm)</li></ul><h4><strong>Conclusions</strong></h4><ul><li>A highly sensitive and robust MRM LC/MS/MS method was developed for the quantitation of N-nitroso duloxetine in duloxetine hydrochloride API and finished formulation using the Agilent 6475A LC/TQ.&nbsp;</li><li>The optimized chromatographic conditions provided effective separation of the analyte from the API, placebo, and formulation matrices, ensuring reliable quantitation.&nbsp;</li><li>The calibration curve was linear over the concentration range of 0.5-40 ng/mL, with 1/x weighting. R and R2 values were observed for both to be greater than 0.99.&nbsp;</li><li>Recovery studies demonstrated efficient sample extraction, with recoveries ranging from 92-99% in API samples and 109-116% in pellet formulations at the LOQ, specification, and 200% specification levels.&nbsp;</li><li>The method also showed excellent reproducibility across all evaluated concentration levels, confirming its suitability for routine analysis.</li></ul><h3><strong>2. Shimadzu / ASMS: Non-Targeted Analysis of Per- and Polyfluoroalkyl Substances in Environmental Extracts by Microflow LC-QToF Coupled with Multi-spray ESI</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/TP_562_Non_Targeted_Analysis_of_Per_and_Polyfluoroalkyl_Substances_in_Environmental_Extracts_by_Microflow_LC_Q_To_F_Coupled_with_Multi_spray_ESI_c2a7e1d5bf.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Targeted screening methods are well established for monitoring Perand polyfluoroalkyl substances (PFAS) in environmental samples. However, PFAS comprise a large and evolving class of compounds, creating a need to screen existing, emerging, and transformation products across diverse matrices. Non-targeted Analysis (NTA) allows for the confirmation of suspected PFAS in the environment, but the sample characterization is often limited by analytical sensitivity. Microflow (0.5 – 40 µL/min) coupled with a multi-nozzle emitter enhances sensitivity by improving desolvation. When this is coupled to a high mass accuracy and resolution Quadrupole Time-ofFlight Mass Spectrometer (QToF), capable of data-dependent and independent MS/MS, PFAS characterization of unknown analytes is possible. In this study, various environmental extracts were processed and screened using NTA for PFAS compounds.</p><h4><strong>Methods</strong></h4><p>A <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=Nexera+Mikros"><strong>Shimadzu Nexera Mikros LC</strong> </a>was used to introduce the sample to a Newomics DuoESI source with an M3 emitter on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/library?mainauthorItems=1&amp;search=9050"><strong>LCMS-9050 QToF MS</strong></a> (Fig. 1). A direct injection on an octadecyl column without delay column was performed. Direct injection was chosen over trapand-elute configuration to minimize selectivity bias caused by the trap column.&nbsp;</p><h4><strong>Conclusion</strong></h4><p>With the increased sensitivity of microflow LC-QToF coupled with multi-spray ESI with DIA acquisition, Insight Explore aided in NTA PFAS analysis. When paired with Insight Discovery and Profiler, a streamlined batch data processing workflow with statistical analysis was shown to effectively handle complex environmental matrices and their analyses.</p><h3><strong>3. Thermo Fisher Scientific / ASMS: Robust and sensitive quantitation of 18 steroids in human serum using Thermo Scientific TSQ Certis Triple Quadrupole mass spectrometer</strong></h3><ul><li>Poster</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/po_004707_lsms_18_steroids_tsq_certis_clinical_po004707_asms2026_na_en_244c52437b.pdf"><strong>Full PDF for download</strong></a></li></ul><p>Accurate quantification of endogenous steroids in human serum is essential for biomonitoring physiological functions such as hormone regulation, reproduction, and immune response in clinical research laboratories. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) is the gold standard for steroid analysis due to its superior sensitivity, selectivity, and multiplexing capability compared to immunoassays. However, steroid measurements remain challenging because of low endogenous concentrations and complex serum matrices. Here, we describe a robust and sensitive LC–MS/MS method for the simultaneous quantification of 18 steroids in human serum using the Thermo Scientific TSQ Certis triple quadrupole mass spectrometer (Figure 1). The method leverages advanced ion source design and fast acquisition to deliver enhanced sensitivity, stability and throughput suitable for high-volume laboratory workflows.</p><h4><strong>Materials and methods</strong></h4><h5><strong>Liquid chromatograph – mass spectrometry&nbsp;</strong></h5><p>Samples were analyzed on a <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/744"><strong>Thermo Scientific Vanquish Horizon UHPLC system</strong></a> coupled to the <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/2207"><strong>TSQ Certis MS</strong></a><strong> </strong>operated in selected reaction monitoring (SRM) mode. The mobile phase composition, analytical column details, LC gradient, and overlaid extracted ion chromatograms (EICs) are shown in Figure 3.&nbsp;</p><h5><strong>Data analysis&nbsp;</strong></h5><p>Data were acquired and processed using <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/77"><strong>Thermo Scientific TraceFinder software</strong></a> (v 5.2).</p><h4><strong>Conclusions&nbsp;</strong></h4><p>A sensitive and robust LC–MS/MS method was developed for simultaneous quantification of 18 steroids in human serum using the Vanquish Horizon UHPLC and TSQ Certis MS with OptaMax Plus ion source. The method demonstrated excellent linearity, accuracy, and reproducibility across a wide dynamic range. Optimization of MS conditions, particularly vaporizer temperature, significantly improved sensitivity. Overall, the workflow delivers high sensitivity, selectivity, and long-term stability, providing a reliable solution for high-throughput steroid analysis in clinical and research settings.</p><h3><strong>4. Waters Corporation: Alkaline-hydrolysis Method for PFAS in Textiles and Textiles Products by Xevo™ TQ-S micro with ACQUITY™ UPLC™ H-Class Plus System</strong></h3><ul><li>Application note</li><li><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/labrulez-bucket-strapi-h3hsga3/720009443_en_0ab4914d72.pdf"><strong>Full PDF for download</strong></a></li></ul><div><div><section><div><div><div><div><div><div><h5><strong>Benefits&nbsp;</strong></h5><ul><li>Alkaline hydrolysis method (EN 17681-1:2025) used for preparing textiles, coated textiles and textile products for PFAS analysis allowed the inclusion of side-chain fluorinated polymers&nbsp;</li><li>Extraction method provided significant advantages in handling complex textile matrices by creating clear extracts free from dispersions, settleable solids and particulates&nbsp;</li><li>Protected the high-value LC-MS/MS components from potential damage&nbsp;</li><li>The method improved analytical sensitivity, reproducibility and accuracy, enabled cost-effective and timeefficient PFAS analysis maintained high analytical quality even for challenging high-matrix samples</li></ul><p>Many PFAS are linked to health concerns because of their ability to interfere with the body’s hormone systems, acting as endocrine disruptors also, linked to other toxic effects. Since textiles come into direct and prolonged contact with the skin, there is an increased risk of human exposure—especially for babies and children.4 In addition, PFAS pose significant environmental concerns. These “forever chemicals” do not readily break down and can be released during manufacturing, washing and disposal, allowing them to enter water pathways and persist in the environment. As a result, testing for PFAS has become increasingly important in the textile industry to demonstrate supply chain transparency, build consumer trust and protect brand reputation.</p><p>Synthetic fluorinated chemicals are categorized into two main groups - non-polymeric PFAS and polymeric PFAS, known for their use as water and grease repellent, heat resistant finishes in textiles, upholstery, leather, apparel, carpet and other industrial applications such as packaging, firefighting foams, non-stick cookware, etc. (Figure 1). PFAS are fluorinated substances contain at least one fully fluorinated methyl or methylene carbon atom (without any H/Cl/Br/I atom attached to it), i.e. with a few noted exceptions, any chemical with at least a perfluorinated methyl group or a perfluorinated methylene group.2 PFAS are classified as long chain and short chain based on their perfluorinated carbon chain length. Short chain fluorinated polymers encompasses a diverse range of chemistries, categorized according to their generic chemical and repeating units structures. Few examples are: derivatives of fluorotelomers, perfluoropolyethers with varying carbon chain lengths and acrylates, epoxylates, urethane, silicones of repeated units based structures.3 Polyfluoroalkyl substances such as fluorotelomer alcohols (FTOHs) contain C–H bonds, rendering them more susceptible to degradation. The ethoxylates in fluorotelomers-methacrylate break down to FTOH whereas polymeric fluorinated urethanes, acrylates break down to PFCAs (non-polymeric and polymeric side chain fluorinated polymers). In contrast, perfluoroalkyl substances such as PFOS and PFOA are extremely persistent due to their fully fluorinated carbon chains.&nbsp;</p><p>In alkaline hydrolysis methods, methanolic aqueous sodium hydroxide is used to break down precursor PFAS such as FTOHs, branched side chain PFAS. This process releases terminal perfluorinated carboxylic acids (PFCAs), which can then be measured. However, the efficiency of hydrolysis varies among different PFAS compounds and the presence of interfering substances such as organic matter or other chemicals in the sample matrix can inhibit the cleavage and detection of these analytes.</p><h4><strong>Experimental</strong></h4><ul><li><strong>LC system:</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/198"><strong>ACQUITY UPLC H-Class Plus System</strong></a> and SMFTN fitted with PFAS kit</li><li><strong>Column(s):</strong> <a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/1823"><strong>XBridge™ BEH™ C18 5 µm Column</strong></a>, 4.6 x 100 mm&nbsp;</li><li><strong>MS system: </strong><a target="_blank" rel="noopener noreferrer" href="https://lcms.labrulez.com/products/209"><strong>Xevo TQ-S micro Mass Spectrometer</strong></a></li></ul><h4><strong>Conclusion&nbsp;</strong></h4><p>By strictly following the critical steps of the alkaline hydrolysis method using methanolic aqueous sodium hydroxide (NaOH) both freely extractable PFAS and those previously bound within side-chain fluorinated polymers such as esters linked to fluorotelomer alcohols (n:2 FTOHs, where n = 4, 6, 8, 10, 12), can be effectively extracted. This approach also safeguards the LC-MS/MS instrumentation from blockages, damage, or contamination by preventing polymer accumulation and matrix interferences.&nbsp;</p><p>The analytical method using the ACQUITY UPLC H-Class Plus System and Xevo TQ-S micro LC-MS/MS System was found to be sensitive, reproducible and accurate, enabling cost-effective and time-efficient PFAS analysis in chemically processed textiles, coated textiles and textile products whilst maintaining high analytical quality even for challenging high-matrix samples.</p></div></div></div></div></div></div></section></div></div>

News from LabRulezLCMS Library - Week 34, 2026

<p><span><strong>This study develops a chiral LC-MS/MS method with pre-column derivatization for simultaneous determination of carnitine (CAR) and acetylcarnitine (ACA) enantiomers in food. Conversion to 3-nitrophenylhydrazine derivatives followed by separation on a CHIRALPAK ZWIX (−) column provided resolution values above 1.57 for the respective enantiomers.</strong></span></p><p><span>After validation, the method was applied to meat, milk, and fermented soybean products. L-CAR and L-ACA were detected in all tested foods, while trace amounts of D-CAR were identified in the traditional Japanese fermented food Hama-natto, demonstrating the method’s suitability for selective food analysis.</span></p><blockquote><h4><strong>The original article</strong></h4><h4><a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#sec0010"><strong>Development of a chiral liquid chromatography-tandem mass spectrometry for simultaneous determination of carnitine and acetylcarnitine enantiomers in food samples</strong></a></h4><p>Aogu Furusho, Hiroki Mizuno, Yutaro Nakayama, Eiji Sugiyama, Kenji Kojima, Kenichiro Todoroki</p><p>J. Pharm. Biomed. Anal. Open., 2026, 100111, Volume 7</p><div><a target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#sec0010">10.1016/j.jpbao.2026.100111</a></div><h6>licensed under <a target="_blank" rel="noopener noreferrer" href="https://creativecommons.org/licenses/by/4.0/"><strong>CC-BY 4.0</strong></a></h6></blockquote><p><strong>Selected sections from the article follow. Formats and hyperlinks were adapted from the original.</strong></p><div><div><div><div><div><p>Carnitine (CAR) is a functional endogenous compound found in various animals <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib1"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[1]</span></a>. Since CAR has one asymmetric carbon in its structure, the <span>l</span>- and <span>d</span>-enantiomers are present. <span>l</span>-CAR is a cofactor that transports long-chain fatty acids into the mitochondria and promotes energy production via β-oxidation <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib2"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[2]</span></a>. Humans biosynthesize 25% of the required amounts of <span>l</span>-CAR from <span>l</span>-lysine and <span>l</span>-methionine and ingest the remaining 75% from foodstuffs <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib3"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[3]</span></a>, <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib4"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[4]</span></a>. The insufficient intake of <span>l</span>-CAR induces a CAR deficiency <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib5"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[5]</span></a>, and levocarnitine (<span>l</span>-form) is used as the therapeutic drug. Acetylcarnitine (ACA) is an acetylated molecule of CAR and is converted from CAR by carnitine <i>O</i>-acetyltransferase in the human body <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib6"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[6]</span></a>. ACA is also related to energy production and is essential for storing acetyl groups to produce acetylcholine <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib7"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[7]</span></a>. A previous study demonstrated that <span>l</span>-ACA taken from foods was partially hydrolyzed in the body and increased the <span>l</span>-CAR concentration within the tissues of aged rats <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib8"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[8]</span></a>, indicating that CAR and ACA are interconverted. Furthermore, <span>l</span>-ACA has some therapeutic effects to downregulate inflammation-induced angiogenesis <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib9"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[9]</span></a> and to reduce nerve pain <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib7"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[7]</span></a>, <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib10"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[10]</span></a>, making it a promising drug candidate. Both CAR and ACA are also commercially available as supplements to reduce body fat.</p><p>Meanwhile, the <span>d</span>-isomers don’t have the above functions and are not thought to be present in the human body. Previous studies have revealed some specific functions such that <span>d</span>-CAR inhibited the <span>l</span>-CAR absorption causing CAR deficiency <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib11"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[11]</span></a>, and <span>d</span>-ACA enhanced the efficiency of the drug for the Pompe disease <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib12"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[12]</span></a>. Since the physiological actions of the <span>d</span>-forms and <span>l</span>-forms differ, enantioselective analysis of the CAR and ACA enantiomers is desirable for studying their functions and biokinetics.</p><p>Several analytical methods have been already developed for the determination of the CAR and ACA enantiomers. In these methods, two approaches were mainly taken; i.e., the reversed-phase separation with the combination of diastereomeric derivatization and the direct separation by an enantioselective column. To convert enantiomers to diastereomers, chiral reagents, such as (+)-1-(9-fluorenyl)ethyl chloroformate <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib13"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[13]</span></a> and <span>l</span>-alanine-β-naphthylamide <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib14"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[14]</span></a>, were used. The conventional ODS column successfully separated the CAR and ACA enantiomers as their diastereomeric derivatives, and the methods were reported to be applicable for the enantiomeric purity check of drug substances. Regarding the chiral liquid chromatographic (LC) method, various types of enantioselective columns have been tested, but it was revealed that the separation of the CAR and ACA enantiomers was quite difficult <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib15"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[15]</span></a>. A CHIRALCEL OD-R column based on the polysaccharide structure was among the few that achieved simultaneous separation of the CAR and ACA enantiomers. The target analytes were derivatized with (α-bromo)methyl phenyl ketone, and the method was applied to the analysis of drug substances <a style="color:rgb(2, 114, 177);font-size:16px;line-height:26px;text-decoration:none;" target="_blank" rel="noopener noreferrer" href="https://www.sciencedirect.com/science/article/pii/S2949771X26000101#bib16"><span style="border-bottom:1.6px solid rgba(0, 0, 0, 0);">[16]</span></a>. However, the measurement of foods, pharmaceutical agents, and biological samples has scarcely been performed, likely due to the low selectivity of the LC separation and ultraviolet/fluorescence detection used in these methods. Thus, improving the selectivity of the method is required to expand the range of the measurable samples.</p><p>In the present study, a chiral LC-tandem mass spectrometric (MS/MS) method has been developed for the simultaneous determination of the CAR and ACA enantiomers. Direct separation using an enantioselective column enables the analysis independent of the optical purity of the reagents, and MS/MS could provide a higher selectivity than those of the previous methods. Furthermore, because CAR and ACA possess an ammonium cation which contributes to the high ionization efficiency, a high sensitivity by MS could also be expected. After the method development and validation, the content analyses of the CAR and ACA enantiomers were performed for meat, milk, and soybean fermented food samples.</p><h4><strong>2. Experimental</strong></h4><h5><strong>2.4. LC-MS conditions</strong></h5><p>The LC-MS/MS system consisted of ultra-high performance liquid chromatographic systems (<a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/en-us/all/products/198"><strong>ACQUITY H-class</strong></a><strong> and</strong><a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/en-us/all/products/199"><strong> I-class</strong></a>, Waters, Milford, MA, USA) and triple quadrupole mass spectrometers (<a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/en-us/all/products/212"><strong>Xevo TQ-S</strong></a><strong> and </strong><a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/en-us/all/products/209"><strong>TQ-S micro</strong></a>, Waters). The chiral separation of CAR and ACA was performed on a CHIRALPAK ZWIX (-) column (3.0 mm I.D.×250 mm, 3 µm, Daicel Corporation, Osaka, Japan) at room temperature. The mobile phases were 5 mM FA/NH4FA in H2O/MeOH/MeCN (A: 2/9/89 and B: 2/19/79, v/v/v) at the flow rate of 0.25 mL/min. The LC gradient property was as follows: 0–25 min, 100% A; 25.1–45 min, 100% B; 45.1–60 min, 100% A. The MS detection was performed with the following conditions: 3.0 kV spray voltage for ESI positive ionization; 150 L/hr cone gas flow rate; 1000 L/hr desolvation gas flow rate; 150°C source temperature; 500°C desolvation temperature. The selected reaction monitoring (SRM) mode was used for the detection, and the set transitions, cone voltage (CV), and collision energy (CE) for the target compounds are summarized in Table 1. The different CEs were used for 3-NPH-ACA on Xevo TQS (10 eV) and Xevo TQ-S micro (20 eV). Data processing was performed using <a target="_blank" rel="noopener noreferrer" href="https://labrulez.com/en-us/all/products/635"><strong>MassLynx v4.2 software</strong></a> (Waters).</p><h4><strong>3. Results and discussion</strong></h4><h5><strong>3.4. Determination of the CAR and ACA enantiomers in food samples</strong></h5><p>The developed method was applied to the analysis of the CAR and ACA enantiomers in food samples. CAR and ACA are known to be abundant in animal-based foods [23], thus, beef, pork, chicken, and milk were measured. The measured cuts of meat were the shank for beef, thigh for pork, and thigh for chicken in this study, and these parts are commonly used in commercially-available ground meats. Fermented foods are reported to contain relatively high levels of d-amino acids, and some of them are considered to be produced by bacteria during the fermentation process [24]. To investigate the production of CAR and ACA by bacteria, three fermented foods made from soy beans (miso, soy sauce, and Hama-natto) were analyzed. Hama-natto is a traditional food from the Hamamatsu City area in Shizuoka Prefecture, Japan. Steamed soybeans are fermented with koji mold, then salted and dried in the sun.</p><p>The chromatograms of the beef and Hama-natto samples are shown in Fig. 4. l-CAR and l-ACA were clearly observed in both samples. The l-forms were also found in the other tested samples, and their quantified values are summarized in Table 4. Comparing the amounts of l-CAR in the meat samples, beef had the highest levels, followed by pork and chicken. The l-ACA levels were also the highest in beef, and chicken contained more than pork.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Pharm_Biomed_Anal_Open_2026_100111_Volume_7_Fig_4_Determination_of_the_CAR_and_ACA_enantiomers_in_beef_56d92bf289.jpg" alt="J. Pharm. Biomed. Anal. Open., 2026, 100111, Volume 7: Fig. 4. Determination of the CAR and ACA enantiomers in beef and Hama-natto samples as their 3-NPH derivatives. The selected ion chromatogram of ACA is shown for 0–30 min, and that of CAR is shown for 30–60 min." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Pharm_Biomed_Anal_Open_2026_100111_Volume_7_Fig_4_Determination_of_the_CAR_and_ACA_enantiomers_in_beef_56d92bf289.jpg 245w," sizes="100vw" width="2247" height="542"></p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Pharm_Biomed_Anal_Open_2026_100111_Volume_7_Table_4_Amounts_of_l_CAR_and_l_ACA_in_the_food_samples_1c2411fe51.jpg" alt="J. Pharm. Biomed. Anal. Open., 2026, 100111, Volume 7: Table 4. Amounts of l-CAR and l-ACA in the food samples." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Pharm_Biomed_Anal_Open_2026_100111_Volume_7_Table_4_Amounts_of_l_CAR_and_l_ACA_in_the_food_samples_1c2411fe51.jpg 245w," sizes="100vw" width="981" height="447"></p><h5><strong>3.5. Confirmation of D-CAR in the Hama-natto sample</strong></h5><p>To confirm the presence of d-CAR in the Hama-natto, four approaches were conducted; i.e., remeasurement of the fraction, detection with a different SRM transition, separation under different LC conditions and use of high-resolution MS. The fraction was placed in a 1.5 mL tube for three min before the MS to purify the d-CAR. The solution (total 750 µL) was dried and dissolved in 50 µL of the mobile phase. The chromatogram obtained by the remeasurement is shown in Fig. 5A, and the result detected by a different SRM transition (297.1 &gt; 102.1) is in Fig. 5B. The peak of d-CAR was clearly observed in both measurements, and the peak shapes were similar. However, the amount of d-CAR was under the LLOQ and could not be quantified. As shown in Fig. 5C, d-CAR was also detected using the different LC conditions described in Section 2.6, suggesting that the peak was definitely derived from the d-CAR. A high-resolution MS analysis detected a molecule with m/z 297.155, which closely matched with the exact mass of 3-NPH-CAR (m/z 297.156). The extracted ion chromatogram of that molecule is shown in Fig. 5D, and the d-CAR peak was clearly observed. These results demonstrated that trace amounts of d-CAR were present in the Hama-natto sample. To the best of our knowledge, this is the first report to find natural d-CAR in foods. One possible origin of this d-CAR is a production by bacteria including koji mold. Some bacteria are known to produce d-amino acids for cell wall formation [32]. The bacteria capable of producing d-CAR may exist and be involved in the fermentation of Hama-natto. Since the l-CAR and l-ACA concentrations in the other fermented foods were lower than that in Hama-natto, there is a possibility to also detect the d-forms from other foods by the improvement of the extraction process. Further research using various food samples is expected.</p><p><img src="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/J_Pharm_Biomed_Anal_Open_2026_100111_Volume_7_Fig_5_Chromatograms_of_the_3_NPH_d_CAR_fractions_2c9fbf4df7.jpg" alt="J. Pharm. Biomed. Anal. Open., 2026, 100111, Volume 7: Fig. 5. Chromatograms of the 3-NPH-d-CAR fractions analyzed by the different analytical methods. The SRM transitions were 297.1 &gt; 102.1 (A) and 145.1 (B). The fraction was analyzed by LC-MS/MS under the different conditions (C) and LC-TOF-MS (D). The detailed analytical conditions are described in Section 2.6." srcset="https://storage.googleapis.com/labrulez-bucket-strapi-h3hsga3/thumbnail_J_Pharm_Biomed_Anal_Open_2026_100111_Volume_7_Fig_5_Chromatograms_of_the_3_NPH_d_CAR_fractions_2c9fbf4df7.jpg 245w," sizes="100vw" width="2663" height="1199"></p><h4><strong>4. Conclusion</strong></h4><p>In the present study, a chiral LC-MS/MS method has been developed for the simultaneous determination of the CAR and ACA enantiomers in food samples. The derivatization with 3-NPH extended the retention time and improved the enantiomeric separations especially for CAR. The validated method was successfully applied to the analysis of meat, milk, and soybean fermented food samples, and trace amounts of d-CAR were discovered in Hama-natto. The present method is able to distinguish enantiomers in various samples and can contribute to clarifying the origin of d-CAR in food samples and searching/developing for new functional food products.</p></div></div></div></div></div>

Development of a chiral liquid chromatography-tandem mass spectrometry for simultaneous determination of carnitine and acetylcarnitine enantiomers in food samples

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