Troubleshooting Analyte Recovery when Using HybridSPE-Precipitation Technology

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Troubleshooting Analyte Recovery when  
Using HybridSPE-Precipitation Technology

Craig Aurand, Charles Mi, Xioaning Lu,  
An Trinh, and Michael Ye

[email protected]

Introduction

HybridSPE™-Precipitation (HybridSPE-PPT) is a new 

sample prep platform for pharmaceutical bioanalysis. The 

technology merges the simplicity of protein precipitation 

with the selectivity of SPE for the selective removal of 

proteins and phospholipids from biological plasma. Removal 

of these two key interferences greatly reduces the risk of 

ion-suppression during LC-MS-MS analysis for improved 

assay sensitivity and reproducibility. In Reporter issues 26.3 

and 26.5, we provided an overview of HybridSPE-PPT and 

offered application examples that illustrate the benefits of 

the technology. In this article, we discuss non-phospholipid 

specific interactions between the HybridSPE phase and the 

sample that could potentially lead to low recovery of certain 

basic and acidic chelator compounds. We conclude our 

discussions with strategies for improving the recovery of 

such problematic compounds. 

How does HybridSPE-PPT work?

When using 96-well HybridSPE-PPT, 100 μL of plasma is 

added to the individual wells followed by 300 μL precipita-

tion agent (1% formic acid in acetonitrile). After a brief 

mixing step to adequately precipitate endogenous proteins, 

vacuum is applied to the plate. As the sample flows 

through the packed-bed/filter-frit assembly, both proteins 

and phospholipids are concurrently removed. Proteins are 

physically removed by low porosity filters whereas phos-

pholipids are chromatographically removed by the station-

ary phase. The resulting eluent is free of both phospholipids 

and proteins, and can be directly analyzed via LC-MS-MS. 

The HybridSPE stationary phase is a patent pending 

zirconia coated silica that is highly selective towards 

phospholipids. Retention is based on a Lewis acid-base 

interaction between the empty zirconia d-orbitals (Lewis 

acid) and the electron pair of the phosphate moiety (Lewis 

base) inherent of all phospholipids. Phosphate is a very 

strong Lewis base and will preferentially interact with 

zirconia over other Lewis bases (Figure 1). 

 The Importance of Formic Acid

Most acidic pharmaceutical compounds contain carboxyl 

(-COOH) groups. When processing acidic compounds using 

HybridSPE-PPT, the HybridSPE Zr-Si stationary phase will 

likely co-retain acidic compounds along with phospholipids, 

resulting in low absolute recovery. To rectify the situation, 

formic acid is added to the precipitation agent and becomes 

part of the sample during HybridSPE processing. Formic acid 

is a stronger Lewis base than most -COOH groups found in 

acidic pharmaceutical compounds. As a result, formate ions 

will tie up the phase’s zirconia ions, minimizing retention of 

acidic analytes of interest. Because formate is not a strong 

enough Lewis base to displace the phosphates, phospholip-

ids preferentially retain on the HybridSPE-PPT phase.

In this application, we 

process three acidic 

compounds (ketoprofen, 

naproxen, and flunixin) and 

two neutral compounds 

using HybridSPE-Precipita-

tion. The analytes were 

spiked into plasma at the 

level of 20 ng/mL. 100 μL  

of plasma was precipitated 

with 300 μL of one of two 

reagents prior to HybridSPE-

PPT: 1) 1% formic acid in 

acetonitrile or 2) neat 

acetonitrile. The resulting 

HybridSPE-PPT eluent was 

analyzed by LC-MS-MS 

(MRM) using an Ascentis 

(continued on page 18)

Figure 1. HybridSPE-PPT 96-well Schematic and Phospholipid Retention Mechanism 

The phosphate moiety of phospholipids is a strong Lewis base (electron 
donor) that interacts with Zr atoms coated on the silica surface.

Proprietary HybridSPE 

Zirconia Coated Silica

The Zr atom acts as a 
Lewis acid (electron 
acceptor) because it 
has empty d-orbitals.

:

Si-OH

O

O

O

O

Zr

Zr

Si-O–

O

Zr+-OH

Upper 5 μm PTFE Frit

Lower 0.2 μm  
Hydrophobic Filter

G004255

G004389

sigma-aldrich.com/hybridspe-ppt

Excerpt from Reporter, Volume 27.2.

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Sample Handling

RP-Amide column. Figure 2 compares the results between 

the two precipitation agents. From the results described in 

Figure 2, complete loss in recovery was observed for the 

three acidic compounds (ketoprofen, naproxen, and 

flunixin) when formic acid was not added to the precipita-

tion agent. When formic acid is added to the precipitation 

agent, greater than 88% absolute recovery was observed 

for each of the acidic compounds (data not shown). In 

contrast, the two neutral compounds, phenylbutazone and 

oxyphenylbutazone were unaffected by the presence of 

formic acid resulting in high recovery under both conditions. 

Troubleshooting Recovery of Chelator and  

Acidic Chelator Compounds

In our research thus far, we have found that certain 

chelator and acidic chelator compounds retain exceptionally 

strong on the Zr-Si stationary phase used in HybridSPE-PPT 

resulting in low 

absolute 

recovery  

(< 40%) when 

using the 

recommended 

primary 

HybridSPE-PPT 

method (100 

μL plasma + 

300 μL formic 

acid in 

acetonitrile). Such chelating compounds can be identified 

as having functional groups with oxygen atoms in the alpha 

and beta positions. To improve recovery, a Lewis base 

stronger than formate is required as a modifier in the 

(continued from page 17)

Figure 2. Comparison of Precipitation Agents (with and 
without formic acid) using HybridSPE-PPT 

Precipitation Agent: 1% formic acid in acetonitrile

Precipitation Agent: neat acetonitrile

precipitation agent. Table 1 lists various Lewis bases and 

their relative retention strength on zirconia. From our 

experience, replacing formic acid with citric acid and adding 

a simple conditioning step can significantly increase the 

recovery of certain chelator and acidic chelator compounds. 

Figure 3 describes specific chelation functional groups and 

lists example compounds with such functional groups. 

Details for the secondary procedure we recommend are 

described in Table 3. By using the secondary procedure 

described in Table 3, recovery for chelator compounds can 

improve from <40% to 65-95%. Mechanistically, citric acid 

is a stronger Lewis base than formic acid, inhibiting 

retention of chelator compounds. However, citric acid is not 

a strong enough Lewis base to displace retention of 

phosphates (i.e., phospholipids). 

Troubleshooting Recovery of Basic and  

Neutral Compounds

Although the primary retention mechanism for HybridSPE 

is based on Lewis acid-base interactions between Zr ions on 

the stationary phase and negatively charged functional 

groups in the sample (e.g., phosphate moiety of phospho-

lipids), secondary interactions derived from the silica surface 

can retain basic and neutral compounds resulting in poor 

recovery. These secondary interactions with silica surface 

include: 1) weak cation exchange and 2) HILIC interactions. 

To disrupt any weak-cation exchange interactions between 

the silica silanol groups (Si-O-) and basic compounds (e.g., 

contains amine functional groups), formic acid should be 

substituted with ammonium formate. The primary procedure 

we recommend uses formic acid as part of the precipitation 

agent. As a result, H+ is the resulting counter-ion used to 

Table 1. Relative Retention Strength  
of Lewis Bases to Zirconia

Relative Retention

  Lewis Base 

Strength on Zirconia

 Hydroxide 

Strongest

 Phosphate 

 Fluoride 

 Citrate 

 Sulfate 

 Acetate 

 Formate 

 Chloride 

Weakest

Figure 3. Low Recovery Chelation Functional Groups  
with Example Compounds

Chelation functional groups that can lead to low HybridSPE-PPT 
recovery and may require citric acid secondary procedure:

Ketoprofen

Naproxen

Flunixin

0 1  2 3 4 5 

Min

0 1  2 3 4 5 

Min

Oxyphenylbutazone

Phenylbutazone

Beta-hydroxy  

caroboxylic  

acids

1,3-Diketone  

type  

compounds

Beta- 

hydroxy  
ketones

1,3-Diol  

type  

compounds

G004760

G004761

G004762

G004764

G004763

G004765

G002553

G003164

Pravastatin

G002555

Atorvastatin

G002554

Fluvastatin

Apigenin

Example compounds with chelation functional groups:

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neutralize exposed silanol groups on the Hy-

bridSPE-PPT phase (Si-O- => Si-OH). For some 

basic compounds, H+ is not a strong enough 

counter-ion to inhibit cation-exchange reten-

tion between silanol groups and basic com-

pounds. This results in poor recovery. By re-

placing formic acid with ammonium formate, 

a stronger ammonium counter-ion (NH

4

+) is 

employed. Ammonium ions are sufficient in counter-ion 

strength to inhibit most (if not all) basic compounds from 

interacting with silanol groups. 

For more polar compounds, secondary HILIC interactions 

(e.g., hydrogen bonding) may occur between basic/neutral 

analytes of interest and the silica surface. These secondary 

HILIC interactions can be disrupted by substituting ac-

etonitrile with methanol as the precipitation agent. To 

further minimize potential secondary HILIC interactions, the 

sample needs to be 25% aqueous prior to HybridSPE-PPT 

processing. Therefore, combining 100 μL plasma with 300 

μL organic precipitation agent is recommended for 

HybridSPE-PPT. For smaller plasma volumes (e.g., 20-50 μL), 

the sample should be diluted with DI water to maintain a 

Table 2. Improvement of Absolute Recovery when Incorporating Ammonium  
Formate in the Precipitation Agent

Standard 

Standard

  Analyte 

 (no matrix) 

Standard 

(no matrix) 

Plasma + 1% 

  (% Absolute  

+ 1% formic 

(no matrix) 

 + 1% NH

4HCO2 

NH

4HCO2

  Recovery) 

acid in MeCN 

+ MeOH 

in MeOH 

in MeOH

 Mirtazapine 

(266/195) 

0.0% 

13.2% 

96.0% 

104.0%

 Risperidone 

(411/191) 

0.0% 

10.4% 

99.1% 

123.3%

 Olanzapine 

(313/256) 

0.0% 

13.6% 

89.4% 

56.4%

final sample volume of 100 μL prior to addition of 300 μL 

precipitation agent. If greater sensitivity is required after 

sample dilution, an evaporation and reconstitution step can 

be added prior to LC-MS-MS analysis. 

In this study, 3 basic compounds experienced low 

recovery when using the primary method (100 μL plasma + 

300 μL 1% formic acid in acetonitrile). 100 μL of spiked  

(20 ng/mL) plasma samples or standard samples (no matrix) 

were precipitated with 300 μL neat methanol, 1% formic 

acid in acetonitrile, or 1% ammonium formate in methanol 

prior to 96-well HybridSPE-PPT processing using the 

“In-well” precipitation method. Absolute recovery was 

assessed by reversed-phase LC-MS-MS (Table 2). From 

these results, significant improvements in HybridSPE-PPT 

recovery of the basic compounds observed when substitut-

ing 1% formic acid in acetonitrile with 1% ammonium 

formate in methanol as the precipitation agent. 

Table 3 summarizes the primary and secondary procedures 

recommended when optimizing conditions for HybridSPE-PPT. 

Conclusion

HybridSPE-PPT technology is a new sample prep platform 

designed for pharmaceutical bioanalysis. The technology 

combines the simplicity of protein precipitation and the 

selectivity SPE by specifically targeting the removal of 

precipitated proteins and phospholipids. The phospholipid 

removal mechanism is based on a Lewis acid-base interac-

tion between the phosphate moiety inherent with all 

phospholipids and the Zr-Si stationary phase. Although a 

primary method that is suitable for most applications is 

available, low recovery can occur. In this report, we 

described the secondary interactions that can take place 

resulting in low recovery and strategies for how to trouble-

shoot these recovery issues. 

For more information and to download the latest 

HybridSPE-PPT instruction sheet, please visit our website  

sigma-aldrich.com/hybridspe-ppt

Table 3. Summary of Recommended Primary and  
Secondary Procedures for 96-well HybridSPE-PPT

Primary Procedure (suitable for 80% of applications):  
Recommended for most applications (basic, neutral, acidic analytes)

  1.  To each well, add 100 μL plasma followed by 300 μL  

1% formic acid in acetonitrile. Mix the sample well  
(e.g., vortex).

  2.  Apply vacuum and collect the resulting eluent for  

LC-MS-MS analysis.

  3.  If low recovery is observed, proceed to Secondary 

Procedures.

Secondary Procedure (acidic & chelator compounds):  
Recommended for low recovery chelator and acidic chelator compounds

  1.  Condition each well with 400 μL 0.5% citric acid in 

acetonitrile (until flow has ceased). 

  2.  To each well, 100 μL plasma followed by 300 μL 0.5% 

citric acid in acetonitrile. Mix the sample well (e.g., vortex).

  3.  Apply vacuum and collect the resulting eluent for 

LC-MS-MS analysis.

Note: 
Recovery of chelator compounds can improve from < 40% to 65-95%
Citric acid is a stronger Lewis base than formic acid inhibiting the retention of chelator 
compounds.
Citric acid is not a strong enough Lewis base to inhibit phosphates (phospholipids)  
from retaining on the HybridSPE phase.

Secondary Procedure (basic & neutral compounds):  
Recommended for low recovery basic and neutral compounds

  1.  To each well, add 100 μL plasma followed by 300 μL  

1% ammonium formate in methanol. Mix the sample well 
(e.g., vortex).

  2.  Apply vacuum and collect the resulting eluent for  

LC-MS-MS analysis.

Note: 
Recovery of basic and neutral compounds can improve from < 40% to > 89%
NH

4

+ (ammonium formate) is a stronger counter-ion than H+ (formic acid) inhibiting 

most basic compounds from interacting with HybridSPE silanol groups (Si-O-).
Methanol is a more polar solvent than acetonitrile further inhibiting any potential 
secondary HILIC interactions between the analyte and HybridSPE silica surface.

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