A Comprehensive Dilute-and-Shoot Method for Creatine and Creatinine in Commercially Available Sports Nutrition Products Using a Single Quadrupole LCMS

Comprehensive dilute-and-shoot LC‑MS method for quantifying creatine and creatinine in commercial sports nutrition products — summary

Significance of the topic

Accurate measurement of creatine and its hydrolysis product creatinine in sports nutrition products is important for consumer safety, label compliance and potential clinical implications. Creatine supplementation is widespread; conversion to creatinine can alter dosage, reduce product efficacy and—because creatinine is a clinical biomarker of renal function—large exogenous inputs may complicate interpretation of serum creatinine measurements. A robust, rapid analytical workflow suitable for routine quality control and stability screening is therefore valuable.

Study objectives and overview

- Develop a simple dilute‑and‑shoot HPLC–single quadrupole MS (LCMS) method for simultaneous separation and quantitation of creatine and creatinine across a broad concentration range.
- Validate linearity, sensitivity, and recovery in multiple commercial product matrices (chewable tablet, gummy, pill, powder, and ready‑to‑drink beverage).
- Assess extraction efficiency, matrix effects and the occurrence of creatinine in products labeled to contain only creatine.

Methodology and sample preparation

- Sample set: commercially available sport nutrition forms — chewable tablet, gummy, pill, powder, and ready‑to‑drink (RTD) beverage. Samples were homogenized and analyzed by dilute‑and‑shoot following dissolution in water.
- Calibration: external calibration range 0.005–100 ppm for both analytes with isotopically labeled internal standards (creatine‑d3 and creatinine‑d3). Calibration weighting 1/C^2; coefficients of determination R2 > 0.9993 (creatine) and 0.9994 (creatinine). Lower limit of quantitation (LLOQ) and limit of detection reported at 0.005 ppm.
- Recovery/accuracy: assessed by standard addition at three spike levels (0.10, 1.00 and 10.0 µg/mL). Reported standard addition recoveries across matrices ranged from ~68.4% to 122%.

Chromatography and MS conditions (key parameters)

- Column and mobile phases:

• Column: Supelco Discovery HS F5 PFP, 3.0 µm, 2.1 × 150 mm.
• Mobile phase A: LCMS grade water with 0.1% formic acid.
• Mobile phase B: LCMS grade methanol with 0.1% formic acid.

- Gradient and runtime:

• Flow: 0.3 mL/min.
• Gradient: 35% B (0–1.5 min) → 95% B (4–5 min) → 35% B (5.01–8.0 min) to include high‑organic clean‑out for carryover mitigation.
• Total run time: 8.0 min; creatine and creatinine baseline resolved with ~0.50 min separation.

- Injection volume: 1 µL.
- Mass spectrometry (Shimadzu LCMS‑2050 single quadrupole; ESI positive):

• Nebulizing gas flow 1.5 L/min; drying gas 3.0 L/min; heating gas 7.0 L/min.
• Desolvation temperature 450 °C; desolvation line temperature 200 °C.
• Interface voltages optimized for signal: ~1.00 kV for creatine, ~0.50 kV for creatinine.

Instrumental setup (separate summary)

- LC system: Nexera HPLC.
- MS: Shimadzu LCMS‑2050 single quadrupole with electrospray ionization (positive mode).
- Column: Supelco Discovery HS F5 PFP, 2.1 × 150 mm, 3.0 µm.
- Internal standards: creatine‑d3 and creatinine‑d3.

Main results and discussion

- Analytical performance:

• Linearity: broad linear range 0.005–100 ppm with excellent R2 (>0.999).
• Sensitivity: LLOQ/LOD at 0.005 ppm for both analytes.
• Precision: calibration/quality criteria within acceptable ranges (signal‑to‑noise >3, %RSD <5% reported for standards/controls).

- Recoveries and extraction efficiency:

• Overall standard addition recoveries across matrices were 68.4–122%, indicating acceptable but matrix‑dependent extraction and quantitation performance.
• Gummy matrix: water extraction yielded 61.5–75.9% recovery, superior to 0.1 N HCl extraction (24.1–30.2%), highlighting that extraction solvent choice critically affects recovery for gelatin/chewable matrices.

- Product content vs. label:

• Measured creatine content (average n=3) showed substantial deviations in some matrices: chewable ~349.4 mg (reported as ~96.4% lower than labeled), gummy ~1181 mg (~5.7% lower than labeled), pill ~791.5 mg (~23.3% lower), powder ~1012 mg (~1.2% higher), RTD ~2857 mg (~6.4% higher). These values indicate product‑specific discrepancies and potential formulation or stability issues.

- Creatinine occurrence:

• Creatinine, not declared on labels, was detected in the chewable (2.26 mg), gummy (46.1 mg) and RTD (1839 mg) samples; no creatinine was detected in the pill and powder matrices. The high creatinine level in the RTD is notable and suggests extensive hydrolysis in that matrix or during storage/processing.

- Method robustness: chromatography allowed rapid separation with an 8‑minute cycle and a high‑organic wash to reduce carryover; MS interface/ionization optimized to maximize signal while minimizing in‑source conversion and suppression.

Practical benefits and applications of the method

- Rapid throughput and minimal sample prep (dilute‑and‑shoot) make the approach suitable for routine quality control, batch release testing and stability screening.
- Single quadrupole LCMS provides a cost‑effective option for laboratories without access to high‑resolution MS while maintaining adequate sensitivity and selectivity when using isotopically labeled internal standards.
- Method can detect unintended creatinine formation, enabling manufacturers to evaluate formulation stability and storage conditions that limit hydrolysis.

Limitations and considerations

- Matrix effects and variable recoveries require careful use of isotopically labeled internal standards and may necessitate matrix‑matched calibration or standard addition for accurate quantitation in challenging samples.
- Some matrices (e.g., gummy) showed low extraction efficiency with acidic solvent; method parameters should be validated per matrix and extraction protocol optimized accordingly.
- Single quadrupole MS provides limited structural confirmation compared with MS/MS or HRMS; coeluting isobaric interferences should be considered in complex matrices.

Future trends and potential applications

- Routine application of dilute‑and‑shoot LCMS methods for product QC and shelf‑life studies to monitor creatine stability and quantify creatinine formation over time and under different storage conditions.
- Extension of the method to stability‑indicating studies, including controlled temperature/humidity stress, to guide formulation improvements for reduced hydrolysis.
- Adoption of orthogonal confirmation (tandem MS or HRMS) for regulatory or investigative testing where structural confirmation of unexpected degradants is required.
- Use of the approach by clinical labs or epidemiological studies to assess exposure from supplements and potential impacts on serum creatinine interpretation.

Conclusions

- A practical, sensitive dilute‑and‑shoot LC‑single quadrupole MS method was developed and demonstrated for simultaneous quantitation of creatine and creatinine in diverse sports nutrition matrices.
- The method delivers rapid analysis (8 min), a wide linear range (0.005–100 ppm), and acceptable sensitivity (LLOQ 0.005 ppm).
- Significant matrix‑dependent differences in recovery and notable deviations between labeled and measured creatine content were observed; creatinine was present in several products labeled to contain only creatine, with especially high levels in one RTD sample. These findings underscore the need for routine QC and stability testing to ensure product quality and to assess potential impacts on clinical creatinine measurements.

References

1. Jaffe A., Gruszecka D., Hain E. R., Monti S., Zerda‑Pinto V., Mendis L. T., Wiest L. A., Gilles C., Ferranti A., Kurzyniec S. A Comprehensive Dilute‑and‑Shoot Method for Creatine and Creatinine in Commercially Available Sports Nutrition Products Using a Single Quadrupole LCMS, WP 337, Shimadzu Scientific Instruments.