Validated quantitation method for a peptide in rat serum using liquid chromatography/high-field asymmetric waveform ion mobility spectrometry

Authors


Abstract

The analysis of peptides presents serious challenges for bioanalytical scientists including low total ion current and non-selective fragmentation during tandem mass spectrometry (MS/MS). During method validation of a peptide in rat serum matrix some interferences could not be easily removed and thus prevented accurate and precise measurement. These problems associated with peptide quantitation were resolved by using FAIMS (high-Field Asymmetric waveform Ion Mobility Spectrometry). This selectivity-enhancing technique filters out matrix interferences, and the resulting pseudo-selected reaction monitoring (pseudo-SRM) chromatograms were nearly free from interferences. Control blank matrix samples contained an acceptable level of interference (only 7% signal as compared to the lower level of quantitation). Chromatographic peaks were easily, accurately and precisely integrated resulting in a validated liquid chromatography (LC)/FAIMS-MS/MS method for the analysis of a peptide drug in rat serum according to United States Food and Drug Administration (US FDA) bioanalytical guidelines. These results confirm that new selectivity-enhancing technologies aid the pharmaceutical industry in reliably producing acceptable pharmacokinetic data. Copyright © 2009 John Wiley & Sons, Ltd.

Peptide bioanalytical method development poses special problems for conventional liquid chromatography/tandem mass spectrometry (LC/MS/MS) systems.1 The total ion current is distributed among several charge states owing to multiple basic ionization sites. Therefore, many multiply charged species are distributed in an envelope of charge states. Even before method development begins, the scientist has less absolute signal than is frequently observed with singly charged small drug molecules.2

Tandem mass spectrometry (MS/MS) offers increased selectivity over single MS, but, because peptide bonds are nearly equal in energy to each other, the ion current becomes distributed among many equally low abundance fragments. To prevent this low response, selected ion monitoring (SIM) is a frequently used option.3 SIM is a sensitive technique due to the high ion current observed, but it is not very selective and hence the popularity of triple quadrupole mass spectrometry. Complex matrices frequently contribute interferences at the m/z for the analyte during SIM.4 These interferences appear as high chemical background and co-eluting chromatographic peaks.5

For compounds that have low ion current due to inefficient fragmentation, one way to take advantage of the selectivity of a triple quadrupole mass spectrometer is to use pseudo-selected reaction monitoring (pseudo-SRM). This experiment involves both mass-resolving quadrupoles at the same m/z with only a small amount of collision energy. The objective is to reduce the response of the interferences preferentially while keeping the peptide intact. In this study, a method was developed for the analysis of a peptide drug by LC/pseudo-SRM. During validation experiments, matrix interferences prevented the successful implementation of the method for analysis of pre-clinical study samples. Thus additional selectivity was required.

FAIMS (high-Field Asymmetric waveform Ion Mobility Spectrometry) increases the selectivity of an MS assay based on the trajectory of an ion in an atmospheric pressure gas.6 This technology has application in small molecule analysis for quantitation as well as qualitative discovery.4, 7, 8 Thus, FAIMS was used in combination with pseudo-SRM at unit resolution to validate a quantitative method for a peptide in rat serum according to good laboratory practices bioanalytical guidelines.9 As this method had already been validated without FAIMS, the novel FAIMS-pseudo-SRM approach was compared against the established assay with respect to linearity, accuracy, precision, lower limit of quantification and selectivity.

EXPERIMENTAL

Chemicals and supplies

Peptide 1 and a 15N-labeled internal standard (IS) were synthesized in house by standard amino acid synthesis methods. Rat serum was purchased from Harlan-Winkelmann (Borchen, Germany). HPLC-grade acetonitrile (ACN), formic acid, trichloroacetic acid (TCA) and trifluoroacetic acid (TFA) were obtained from commercial sources and used without further purification. Industrial grade gases and deionized water were used throughout.

Sample preparation

Stock solutions of peptide 1 and IS were prepared by accurately weighing the materials and dissolving in TCA 0.02% (m/m). Calibration (standard) and validation (quality control) samples were prepared by adding appropriate volumes of peptide 1 stock solution into control rat serum. All solutions were divided into aliquots and stored at or below −18°C until use. To each aliquot (100 µL) was added 20 µL IS working solution. Samples were processed by the addition of 120 µL of TCA (3.5%, m/m), by shaking on a laboratory shaker for 60 s, and by centrifugation at 12 000 rpm for 10 min. Finally, the supernatant was transferred into sample vials for injection onto the LC system.

At the onset of each measuring sequence a set of eight calibration samples at six non-zero concentrations was measured. The lower level of quantitation (LLOQ) and the upper level of quantitation (ULOQ) were processed in duplicate. The calibration standards were adequately distributed over the whole calibration range (10.0, 30.0, 100, 249, 1000 and 1998 ng/mL). Additionally, one matrix sample (blank) and one matrix sample fortified with IS (zero) was analyzed within every batch.

Validation samples were prepared at the following concentrations: the LLOQ (10.1 ng/mL), a low concentration (between the LLOQ and 2–3 times that of the LLOQ, 29.6 ng/mL), the mid-range concentration (between 30% and 60% of the ULOQ, 499 ng/mL) and the ULOQ (1995 ng/mL).

Instrumental

The LC system consisted of an 1100 series binary pump and degasser (Agilent Technologies, Santa Clara, CA, USA) and a HTC-PAL autosampler (CTC Analytics, Zwingen, Switzerland). The mobile phases were composed of A: 0.1% TFA in water, and B: 0.1% TFA in ACN, at a total flow rate of 0.2 mL/min. The mobile phase gradient proceeded according to the following scheme. After injection, mobile phase B was held at 5% for 1 min. Then, it was linearly increased to 60% over 6 min. After 7.5 min mobile phase B was increased to 80% and held for 2.5 min. Mobile phase B was then immediately reduced to 5% for column re-equilibration. The total run time was 12 min. Twenty microlitres of sample were injected. The column used was an Atlantis dC18 (30 × 2.1 mm, 3 µm; Waters Corporation, Milford, MA, USA).

The FAIMS system (Thermo Fisher Scientific, San Jose, CA, USA) used the recommended standard conditions of dispersion voltage (DV) −5 kV, 4 L/min equimolar nitrogen and helium, inner electrode temperature 70°C and outer electrode temperature 90°C. A reference solution of peptide 1 was infused at 5 µL/min and combined via a mixing tee with 50% mobile phase B flowing at 0.2 mL/min. The compensation voltage (CV) was ramped and the voltage at which the maximum signal appeared was the experimentally determined CV. The resulting CV for peptide 1 and IS was −35 V. Optimum response was achieved by ensuring the probe position was set to the furthest back possible (standard conditions). This provided for sufficient desolvation which in turn translated to a stable maximum signal.

The triple stage quadrupole (TSQ) mass spectrometer was a TSQ Quantum Ultra controlled by LCquan 2.5 software (Thermo Fisher Scientific, San Jose, CA, USA). Positive mode heated electrospray ionization (H-ESI) was used with the following parameters: ion spray voltage, 4000 V; vaporizer temperature, 400°C; tube lens offset, 228 V. Optimized pseudo-SRM conditions for peptide 1 included m/z 1277.3 and 1277.3 for Q1/Q3 masses and 25 eV collision energy. For the IS, the Q1/Q3 masses were 1292.7 and 1292.7, with a collision energy of 25 eV. Both mass-resolving quadrupoles were set to unit resolution (0.7 u FWHM). The m/z values correspond to the quintuply charged [M–H]5+ ions of peptide 1 and the IS. Collision gas pressure was set to 3.0 mTorr.

Data treatment

Integration of the chromatographic peaks was performed by LCquan 2.5. Concentration was determined by plotting peak area ratio vs. nominal concentration and back-calculating area ratios from the regression line using a weighting factor of 1/x2. All statistical calculations were performed using Microsoft Excel Office 2003 (Microsoft Corporation, Redmond, WA, USA). All concentrations are reported with three significant figures except for integer values and numbers greater or equal to 1000. Precision was assessed by the coefficient of variation (CV%) calculated from the measured concentrations. Accuracy (bias %) was calculated as the relative difference between the mean measured and the nominal concentrations.

Method validation criteria

Calibration samples were rejected from the calculation of the response function if the deviation from the nominal concentration exceeded 15% (20% at the LLOQ). At least one sample at the LLOQ and ULOQ had to meet this specification and could not be rejected. If more than 25% of the calibration samples failed then the analysis batch had to be rejected. The precision and accuracy of the method was evaluated after replicate analysis (n = 6 at each concentration level) of the validation samples. The precision had to be less than or equal to 15% (20% at the LLOQ) and the accuracy had to be within ±15% (±20% at the LLOQ).

Selectivity at the LLOQ

For the confirmation of the LLOQ the mean peak area of six zero samples (control rat serum of six different origins fortified with IS) was compared to the mean peak area found in six LLOQ validation samples prepared from the same source serum as the zero samples. The mean peak area of the zero samples should not exceed 20% of the mean LLOQ peak area.

RESULTS AND DISCUSSION

Attempts to fragment this multiply charged peptide and use an SRM transition with different m/z ratios, and thus fully utilize the selectivity of the triple quadrupole mass spectrometer, resulted in no significant single fragment being formed. Thus, LC/pseudo-SRM analysis resulted in chromatograms with abundant signal but numerous background peaks at the retention time of the peptide. The use of highly selective reaction monitoring (H-SRM), which is a high-resolution MS experiment, resulted in no improvement to the signal-to-noise (S/N) ratio. Figure 1 shows a representative chromatogram at the LLOQ. The LLOQ may seem rather high at 10 ng/mL, but for this peptide drug there was no need to develop the method to lower range. The unknown sample concentrations (therapeutic range) were expected to be within the limits used.

Figure 1.

Representative LC/MS chromatogram for the analyte and IS at the LLOQ. The upper trace (analyte) shows a signal-to-noise ratio of 303, a raised baseline, and significant interferences. The lower trace (IS) shows a peak for the IS at the same retention time as the analyte, also with raised baseline.

A peak was detected and integrated at approximately 6.6 min, but there were closely co-eluting interferences that adversely affected the quality of the integration. Likewise, the internal standard (IS) demonstrated a raised baseline indicating chemical background. The interferences appeared to be multiply charged species.

Despite these observations, the assay passed the initial validation criteria using this method as shown in Tables 1–3 (Without FAIMS results). However, during confirmation of selectivity at the LLOQ, serum demonstrated many interferences, as shown in Fig. 2 and quantified in Table 4 (Without FAIMS results). Blank samples demonstrated chromatographic peaks at the same retention time as the analyte, despite no analyte being added to the samples. According to bioanalytical guideline9 criteria, blank matrix samples should have less than 20% of the LLOQ analyte signal. By LC/MS alone, control serum appeared to have as much as 45% and on average approximately 35% of the LLOQ signal. The interference signal was not due to endogenous analyte because of the synthetic, exogenous nature of the peptide. It was for these reasons that FAIMS was explored as a solution to the interference problem.

Table 1. Calibration standard samples without and with FAIMS. There is essentially no difference between the %-deviation from theoretical
  Nominal concentration (ng/mL)
  10.030.010024910001998
Without FAIMSReplicate 110.329.91052509771917
Replicate 29.672032
Average9.9829.91052509771974
Accuracy (%)−0.204−0.2255.070.277−2.35−1.18
With FAIMSReplicate 19.6229.010225710271861
Replicate 210.52029
Average10.029.010225710271945
Accuracy (%)0.396-3.42.053.102.72−2.65
Table 2. Calibration regression statistics without and with FAIMS. The slope of the regression line with FAIMS appeared higher than without FAIMS, suggesting greater detector sensitivity to changing concentrations. With FAIMS, a lower y-intercept confirms that fewer interferences obstructed accurate and precise quantitation. The coefficients of determination were similar for both analyses
 SlopeInterceptCoefficient of determination (r2)
Without FAIMS0.00700.03700.999
With FAIMS0.01090.02480.998
Change56%−33%
Table 3. Validation levels for quality control. Precision was comparable for both methods, but accuracy was better using FAIMS
 Nominal concentration (ng/mL)
 10.129.64991995
Without FAIMS8.1426.35082015
8.4429.74882026
10.328.14702085
7.6526.84822061
9.0229.14582033
10.324.94882089
Average8.9627.54822051
RSD (%)12.36.593.581.53
Accuracy (%)−11.3−7.19−3.352.83
With FAIMS9.4430.95442057
10.529.75591958
8.8427.85141995
8.5030.35121977
9.0028.45382046
10.829.65371990
Average9.5129.45342004
RSD (%)9.733.953.421.96
Accuracy (%)−5.87−0.5737.030.439
Figure 2.

Representative LC/MS chromatogram of peptide 1 for the analysis of a blank sample in control matrix. Despite no analyte being added, a signal appears at the retention time for the analyte. This demonstrates that SIM is not a very selective analysis technique. Note that SRM resulted in selective transitions, but very low sensitivity due to no abundant favored transition (data not shown).

Table 4. Confirmation of LLOQ (10 ng/mL) experiment. Without FAIMS blank serum extracts showed greater than 20% of the LLOQ signal. Because FAIMS could remove the interferences, the remaining signal is only 7% of the LLOQ. This remaining signal could be attributed to unlabeled material present in the synthetically prepared IS or carryover
 Matrix lotBlankLLOQ% of LLOQ
 178623128968127.1%
 210551129804335.4%
Without FAIMS312018735895933.5%
 49687628255034.3%
 514740432279745.7%
 610707733479832.0%
 Average10928031447134.8%
 1354053692229.6%
 2344063915898.8%
With FAIMS3168123713754.5%
 4161913207915.0%
 5164703232185.1%
 6303163576128.5%
 Average249333556357.0%

Figure 3 shows a representation of the FAIMS electrodes. The electrodes are shown in cut-away form to display the inner electrode and the analysis gap between the inner and outer electrodes. Ions generated by the H-ESI spray emitter enter the FAIMS device through the ion entrance and are analyzed in the mixture of helium and nitrogen between the electrodes. After analysis, ions with a stable trajectory leave the FAIMS device through the ion exit and enter the high-vacuum region of the mass spectrometer through the MS inlet. The FAIMS device acts as a filter because interfering ions do not have a stable trajectory through the device and are removed from the ion beam.10

Figure 3.

Perspective cutaway-view of FAIMS electrodes (center), the MS inlet (left), and H-ESI source (right). The cutaway of the FAIMS electrodes shows the inner and outer electrodes. During FAIMS, ions are separated in the gap between these electrodes. The separation occurs in the presence of a FAIMS carrier gas, in this case a mixture of 50:50 helium and nitrogen.

Standard conditions were used as a starting point for investigating the behavior of peptide 1 and the IS in FAIMS. After 1 h of experimentation, the standard conditions were demonstrated to be the best and so were used without further change. In Fig. 4, representative LC/FAIMS-MS chromatograms demonstrated that the chemical background is greatly reduced versus the without FAIMS chromatograms (Fig. 1). Fewer co-eluting peaks appear in both the analyte and IS transitions. The S/N ratio was improved by 18 times at the LLOQ.

Figure 4.

Representative LC/FAIMS-MS chromatogram for the analysis of the analyte (upper trace) and IS (lower trace) at the LLOQ in serum at CV = −35 V. The signal-to-noise ratio is 5677, which is 18-fold better than the experiment without FAIMS.

A visual indication that control blank chromatograms with FAIMS contained fewer interference peaks is shown in Fig. 5. Analyte traces are normalized to each other (Figs. 5(A) and 5(C)) as are IS traces (Figs. 5(B) and 5(D)). Figures 5(B) and 5(D) confirm that the IS response is essentially identical for the with and without FAIMS experiments, respectively. The interferences in Fig. 5(C) are removed when using FAIMS (Fig. 5(A)).

Figure 5.

Representative LC/FAIMS-MS (A, B at CV = −35 V) and LC/MS (C, D) chromatograms for the analysis of a blank sample in control matrix. The analyte interferences in (C) have been removed by using FAIMS (A). For response comparison, the IS response for LC/MS (D) is essentially identical with LC/FAIMS-MS (B).

Despite the selectivity that FAIMS offers, there is still a trace response in the analyte channel (Fig. 5(A)). The resulting interference level was 7% of the LLOQ. This remaining response is likely due either to residual unlabeled peptide present in the synthetic preparation of the IS or due to autosampler carryover. Further reduction of this contribution might be achieved either by improved peptide synthesis and purification of the IS or by enhanced autosampler washing techniques.

Apart from this dramatic improvement in S/N the performance of the FAIMS assay was comparable or even better than that of LC/pseudo-SRM. The back-calculated concentrations of calibration samples with and without FAIMS resulted in essentially no difference in the %-deviation from the theoretical values, as shown in Table 1. Linear regression in Table 2 revealed a significantly higher slope with FAIMS (0.0109 with and 0.00699) than without FAIMS, respectively. This suggests that changing concentrations of analytes results in greater detector sensitivity when using FAIMS. With FAIMS, a lower y-intercept was obtained (0.0248 with and 0.0370 without FAIMS, respectively) confirming that fewer interferences obstructed accurate and precise quantitation. The coefficient of determination was approximately identical for both techniques.

Validation samples give a measure of assurance that the calibration samples have been prepared and extracted correctly. Table 3 shows that accuracy of these validation samples was better with FAIMS (7.03% difference) than without FAIMS (11.3% difference). The precision of the assay, as defined by the relative standard deviation (RSD) of these validation samples, was comparable for both with FAIMS (9.73% RSD) and without FAIMS (12.3% RSD).

One active area of interest in FAIMS research is the tendency to lower response during analysis.11 By comparing the IS area response with and without FAIMS, the modulation of response could be quantified. Within each experiment type the IS response was consistent. Table 5 demonstrates that FAIMS reduces the chromatographic peak area to approximately 90% of the non-FAIMS experiment similar to previously published results.4 This represents a change within the experimental error of the among-sample variation in the IS response.

Table 5. Chromatographic peak areas for peptide 1 calibration standards. At low concentration fewer interferences are transmitted into the mass spectrometer. At higher concentrations the more important effect is the focusing of ions which results in an increased response relative to the without FAIMS experiment
SampleArea response of peptide 1Deviation [%]
No FAIMSFAIMS
K1029060333101612
K10305058344303
K3074646393522720
K1002246127293356023
K2504968841700393929
K1000201169172908069231
K2000418250475642959932
K20003968501662691691
SampleArea response of ISDeviation [%]
No FAIMSFAIMS
K1027749152383436−11
K1027986332654125
K3030280242742534−10
K10029078882578048−13
K25027842342479382−12
K100029273552589650−13
K200031094192776617−5
K200027829002829136

A second interesting observation on the sensitivity of analysis with FAIMS is described in Table 5. The chromatographic peak area at the LLOQ (sample K10, 10 ng/mL) in the with FAIMS experiment is enhanced 12% relative to the without FAIMS experiment. At higher concentrations the chromatographic peak area further increases relative to non-FAIMS. This observation results in the 56% higher calibration line slope with FAIMS as calculated in Table 2.12

The IS response was consistent within each experiment type. A comparison of IS peak areas as performed in Table 5 demonstrates that FAIMS reduces the signal to approximately 90% of the non-FAIMS experiment. This result seems to contradict the response-enhancing effect of FAIMS for peptide 1. It could, however, be explained by the removal of co-eluting interfering signals lying underneath the internal standard peak by FAIMS.

CONCLUSIONS

Peptide quantitation according to GLP guidelines was achieved using LC/FAIMS-MS. Endogenous interferences which prevented validation in a traditional LC/MS assay were successfully removed. The resulting chromatographic cleanup was characterized by an 18-fold increase in chromatographic S/N ratio and an acceptable confirmation of the LLOQ experiment. FAIMS is a useful bioanalytical tool and an investment in developing robust, reproducible methods.

Ancillary