An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of levetiracetam in human serum and plasma

Chemicals and reagents

LC-MS grade reagents methanol (CAS 67-56-1) and formic acid (CAS 64-18-6) were purchased from Biosolve (Valkenswaard, The Netherlands). Dimethyl sulfoxide (DMSO) (CAS 67-68-5, ACS reagent, ≥99.99%), ammonium acetate (CAS 631-31-8, LC-MS grade), isopropanol (CAS 67-63-0, high-performance liquid chromatography [HPLC] grade), and levetiracetam (CAS 102767-28-2, Cat. No. PHR1447, Lot LRAC2807) were obtained from Sigma-Aldrich (Taufkirchen, Germany). The internal standard [²H₆]-levetiracetam (CAS 1133229-30-7, Cat. No. C597, Lot ES-ALS-14-088-P2) was purchased from Alsachim (Illkirch-Graffenstaden, France). DMSO‑d₆ (CAS 2206-27-1) and qNMR internal standard 1,3,5-trimethoxybenzene (TraceCert^®, Lot Nr. BCBW3670, CAS 621-23-8) were obtained from Sigma-Aldrich Germany.

Native human serum (Art. No. S1-LITER) was obtained from Merck (Darmstadt, Germany), TDM-free human serum (multi-individual pooled; surrogate matrix, ID No. 12095432001) from Roche Diagnostics GmbH (Mannheim, Germany), native plasma matrix (lithium-heparin [Li-heparin], K₂-EDTA and K₃-EDTA) from anonymized, residual patient samples, and water was purified using a Millipore Milli-Q 3 UV system from Merck (Darmstadt, Germany). Pools were prepared from the patient samples in accordance with the Declaration of Helsinki.

qNMR for determination of the purity of the standard materials

qNMR measurements were performed on a JEOL 600 MHz NMR spectrometer with a He-cooled Cryoprobe. Single-pulse 1H{13C} NMR (Supplemental Material 2, Figure 1) was utilized for the quantitation. Additional details about qNMR acquisition and FID (free induction decay) processing parameters can be found in Supplemental Material 2.

Preparation of calibrators and quality control (QC) samples

In brief, two calibrator stock solutions were prepared for the subsequent preparation of spike solutions and the final matrix-based calibrator levels. Per stock solution, 90 mg levetiracetam was weighed on a micro balance (XPR2, Mettler Toledo) and dissolved in 5 mL DMSO in a volumetric flask to produce concentrations of 18.0 mg/mL. The concentration of each primary stock solution was calculated based on the purity of the reference material (99.9% ± 0.1%, determined by qNMR) and the amount of levetiracetam added.

Each primary stock solution was used to prepare working solutions of 1.80 mg/mL, from which calibrator spike solutions of different concentrations were obtained. Final matrix-based calibrators (eight levels [Cal 1–8]), uniformly distributed from 1.53–90.0 μg/mL (8.99–529 μmol/L), (Figure 1) were prepared by a 1 + 99 dilution (v/v) into human serum matrix.

Figure 1:

Schematic overview of levetiracetam calibrator and control levels chosen to allow optimal coverage of measurement and therapeutic reference ranges. Black circles, calibration samples 1–8; black triangles, QC levels 1–4; black diamond, alert level; black line, measurement range; dotted black line, therapeutic reference range. Conversion factor µg/mL to µmol/L: 5.9.

Four levels of matrix-based QC samples (QC levels [QC 1–4]) were prepared in the same way as the calibrator levels, using an independent third primary stock solution. The concentrations for the control levels were set at four critical points: above the limit of quantitation, below and within the therapeutic range, and at the laboratory alert level (Figure 1). Final concentration levels were 2.40, 7.50, 20.0 and 60.0 μg/mL, respectively. All samples (spiked and native material) were stored at −20 °C for a maximum of 27 days. All liquids were allowed to reach room temperature prior to use.

Internal standard (ISTD) solution

For the preparation of the ISTD stock solution, [²H₆]-levetiracetam was dissolved in DMSO to obtain a 1,000 μg/mL solution and stored at −20 °C for a maximum of 6 months. The ISTD working solution was prepared by a twofold dilution of the ISTD stock solution (40 µL DMSO added to 60 µL ISTD stock solution) followed by the addition of 3,900 µL Milli-Q water to achieve a final concentration of 15 μg/mL.

Sample preparation

ISTD working solution (100 µL) was transferred into a 2 mL tube (Eppendorf Safe-Lock Tubes) and 50 µL of the sample specimen (either native sample, calibrator or QC material) were added. To ensure an ISTD equilibration, the sample was mixed briefly on a vortex and then incubated for 15 min (500 rpm, 37 °C) on a ThermoMixer^® (Eppendorf, Hamburg, Germany). Proteins were precipitated by adding 75% methanol (v/v) followed by an incubation on a thermomixer for 10 min (2000 rpm, 23 °C). Then, proteins were separated by centrifugation at 4 °C and 20,000 rcf for 10 min. Subsequently, 10 µL supernatant were diluted 1 + 99 (v/v) using mobile phase A in an HPLC vial (Wicom, Heppenheim, Germany).

Liquid chromatography-mass spectrometry (LC-MS)

Chromatographic separation was performed by an Agilent 1290 Infinity II LC system (Santa Clara, California, USA) equipped with a binary pump, a vacuum degasser, an autosampler at 7 °C, and a column compartment tempered to 40 °C. A Waters SymmetryShield RP8 (100 × 3 mm, 3.5 µm, Milford, Massachusetts, USA) was used for chromatography. Mobile phases consisted of 2 mM ammonium acetate in Milli-Q water with 0.1% formic acid (A) and methanol:2 mM ammonium acetate in Milli-Q-water 95:5 (v/v) with 0.1% formic acid (B).

Measurements were performed at a flowrate of 0.6 mL/min within a total runtime of 10 min. The injection volume was 5 μL. Contamination of the mass spectrometer was reduced using a divert valve to switch the eluent flow until 0.6 min and from 7.0 min to the waste.

Levetiracetam was detected in multiple reaction monitoring mode using an AB Sciex Triple Quad 6500+ mass spectrometer (Framingham, Massachusetts, USA) with a Turbo V ion source operating in positive electrospray ionization (ESI) mode. An ion spray voltage of 4,500 V and a temperature of 300 °C were applied. Curtain gas, collision gas, and ion gas source 1 and source 2 were set at 35, 8, 55 and 55 psi, respectively. For all mass transitions, a declustering potential of 65 V and a dwell time of 20 ms were applied. Several analyte specific mass transitions were tested and optimized regarding intensity and reproducibility resulting in following transitions: m/z 171.1 → 69.1 (quantifier) and m/z 171.1 → 98.0 (qualifier). Monitoring the quantifier/qualifier ratio in native matrix samples compared to the quantifier/qualifier ratio of neat SST samples allowed checking for interfering substances within matrix samples.

Table 1 shows an overview of the selected reaction monitoring transitions and the remaining compound-dependent MS settings.

System suitability test (SST)

An SST was performed prior to each analysis to check the sensitivity and chromatographic performance of the system. Concentration levels of SST1 and SST2 corresponded to the analyte concentration within the processed calibrator levels 1 and 8, respectively. To pass the SST, a signal-to-noise ratio of the quantifier transition of ≥10 for SST1 and retention time of 2.9 ± 0.5 min for SST1 and SST2 were required. Carry-over effects were examined by injecting the high-concentration sample SST2, followed by two solvent blanks. In addition, the analyte peak area observed in the first blank after the injection of the SST2 sample must be ≤20% of the analyte peak area of SST1 or calibrator level 1.

Calibration and structure of analytical series, and data processing

The assay was calibrated using the calibrators described in section “Preparation of calibrators and quality control (QC) samples”. The calibration curve was generated from the area ratios analyte/ISTD for calibrators 1–8 and using a quadratic fit with a 1/x² weighting. The curve was not forced through the origin. For processing of the raw data file, Analyst^® software (version 1.6.3 or higher) was used with the IntelliQuant algorithm. The levetiracetam and [²H₆]-levetiracetam signals showed a retention time of 2.9 min and were integrated within a 30 s window. Peak integration included a smoothing factor of 3 and a peak splitting factor of 3. The noise percentage was set to 90% with a base sub-window of 30 s.

Method validation

Assay validation and determination of measurement uncertainty were performed according to the Clinical & Laboratory Standard Institute Guidelines C62A Liquid Chromatography-Mass Spectrometry Methods [26], the International Conference on Harmonization guidance document Harmonised Tripartite Guideline Validation of Analytical Procedures: Text and Methodology Q2 (R1) [27], and the Guide to the Expression of Uncertainty in Measurement [28].

Traceability to SI units

Regarding traceability to the SI-unit kilogram, a qNMR ISTD directly traceable the primary standard NIST PS1, has been applied for qNMR measurements [23]. Sixfold experiments (Supplemental Material 2, Figure 2), involving six individual weightings of the analyte and the qNMR ISTD, yielded for the calibrator material a final levetiracetam content value of 99.9 ± 0.1% (k=1).

Selectivity

The developed solvent gradient combined with the reversed-phase column (Waters SymmetryShield RP8, 100 × 3 mm, 3.5 µm) allowed to place levetiracetam at a retention time of 2.9 min well separated from polar and apolar matrix components (Figure 2). Selectivity was determined by analyzing sample pools of analyte-free human serum, surrogate serum, and human Li-heparin plasma. No signals were observed at the expected retention time. Moreover, no levetiracetam signal was detected in the [²H₆]-levetiracetam ISTD ion trace.

Figure 2:

Levetiracetam LC-MS/MS derived analytical readouts. (A) Chromatogram of a matrix blank showing the analyte SRM ion trace (left) and the ISTD SRM ion trace (right). (B) Chromatogram of the lowest calibrator level with a concentration of 1.53 μg/mL spiked in native serum; analyte (left) and ISTD (right). (C) Pooled patient sample with a concentration of 36.5 μg/mL; analyte (left) and ISTD (right).ISTD, internal standard.

Measuring native patient samples within the method comparison study no interferences were observed in the retention time window of levetiracetam. In a few samples a peak occurred at 4.9 min. However, this peak does not interfere with the analyte.

Matrix effect

High dilution after protein precipitation, as detailed in the protocol, minimized matrix-dependent effects caused by salts, proteins, and phospholipids. No suppression or enhancement occurred at the expected retention time of levetiracetam independent from the matrix.

Slopes and coefficients of determination of calibrators in different matrices were compared. Slopes were found to be 0.0512 (95% CI 0.0509 to 0.0515) for native serum matrix, 0.0513 (95% CI 0.0508 to 0.0518) for neat solution, 0.0507 (95% CI 0.0503 to 0.0510) for surrogate serum matrix, and 0.0507 (95% CI 0.0504 to 0.0511) for plasma matrix. Correlation coefficients were 1.00 independent of the matrix used for calibration. Furthermore, calibrator samples in surrogate serum, native serum, and Li-heparin plasma were evaluated as controls (n=6 sample preparations) by applying the neat calibration as standard. Relative bias for all matrices and levels ranged from −1.6 to 1.5% with coefficients of variation (CVs) of less than 2.4%.

Additionally, matrix effects were determined according to Matuszewski et al. [30]. No matrix effect was observed for all levels, with mean values of 95–104% for the analyte and the corresponding ISTD (Table 2). The mean area ratios were 99–100% with the 95% CI interval ranging from 97–102%. Thus, the compensating effect of the labelled ISTD was confirmed.

Linearity

Linearity was determined by analyzing six native serum calibration curves, which were adjusted to extend the desired measuring range by 20% at both lower and higher concentrations. The residuals were randomly and equally distributed in a quadratic regression model. The correlation coefficients were r=1.00 for all individual calibration curves.

The linearity of the method was confirmed using serially diluted samples. The measurement results showed a linear dependence with a correlation coefficient of 1.00. The relative deviation (n=6) ranged from 0.4 to 3.0% and the CV was ≤1.5%.

Lower limit of measuring interval (LLMI) and limit of detection (LOD)

The LLMI was determined using spiked samples at the concentration of the lowest calibrator level (1.53 μg/mL). Relative bias showed a deviation of 0.7% while the CV was 2.2%. The LOD was estimated as 0.130 μg/mL with an intensity corresponding approximately to one tenth of the average calibrator 1 peak height.

Accuracy and precision

For the validation of precision and accuracy, spiked serum and plasma samples at four concentration levels were used. Each sample was prepared sixfold from two different operators on five different days. Prior to sample preparation, two high-concentration samples were diluted with native blank matrix and sample preparation performed as described.

The evaluation of precision was performed in a multi-day validation experiment. Variability components between injections, between preparations, between calibrations, and between days were determined using an ANOVA-based variance component analysis to assess the overall variability of the methods. Dependency of intermediate precision, including variances as between-day -calibration, -preparation, and -injection from the matrix, was <2.2%. Repeatability CV showed a range of 1.1–1.7% across all concentration levels; patient sample measurement variability was found indistinguishable from spiked materials (Table 3). The evaluation of the trueness within this experimental setup was evaluated by comparing the measured data to calculated sample concentrations. The mean bias (n=6) showed slightly negative results for samples in serum and plasma, ranging from −2.5% to −0.3%, whereas the mean bias of diluted samples ranged from −0.1 to 2.9% (Table 4). The found deviations from zero are within the range of the measurement uncertainty. Hence, within the limitations of this experiment, no conclusion can be drawn, if the found numbers are deviating from zero are true systematic errors. Consequently, the total measurement uncertainty of individual measurement campaigns is understood as acceptance goal of this procedure.

Stability

Autosampler stability of processed samples (7 °C) was demonstrated for 6 days. The recovery was 96–101%. The stability of pure spike solutions and spiked control samples (−20 °C) was 27 days, and the recovery was 100–106% compared with the original value.

Equivalence of results between independent laboratories

The 130 samples (native serum and plasma patient samples, patient pools, and spiked samples) were analyzed at Sites 1 and 2. Two samples were outside the calibration range (one above and one below), therefore they were excluded from the analysis. A repeated measurement by diluting high concentrated sample was not possible since it was a leftover sample, and the volume was not sufficient. Passing–Bablok regression analysis showed very good agreement between the two laboratories and resulted in a regression equation with a slope of 1.00 (95% CI 0.99 to 1.00) and an intercept of 0.0208 (95% CI –0.11 to 0.14) (Figure 3A). The Pearson correlation coefficient was found to be ≥0.999. The relative Bland–Altman analysis plot also showed a good agreement between the two laboratories (Figure 3B). The data scatter is independent from the analyte concentration with a 2S agreement of 6.5% (lower limit CI interval from −7.6 to −5.6%, upper limit CI interval from 5.4 to 7.4%). Since the performance evaluation in Site 2 yielded an intermediate precision comparable to Site 1 (better than 3.0%), the data scatter was in good agreement with the error propagation between the two independent measurements. The result bias in the patient cohort was −0.1% and not statistically significantly different from zero (95% CI interval from −0.7 to 0.5%). Hence, both data scatter and data bias indicate that the proposed levetiracetam RMP is transferable between independent laboratories.

Figure 3:

Results from the patient sample-based levetiracetam method comparison study performed between two independent laboratories. (A) Passing–Bablok regression plot including the Pearson regression analysis for the method comparison study of the RMP (n=128 patients) between the independent laboratories (Site 1: Risch; Site 2: Roche). Passing–Bablok regression analysis resulted in a regression equation with a slope of 1.00 (95% CI 0.99 to 1.00) and an intercept of 0.0208 (95% CI –0.11 to 0.14). The Pearson correlation value was ≥0.999. (B) Bland–Altman plot for the method comparison study of the RMP (n=128 patients) between two independent laboratories (Laboratory 1: Risch site, and Laboratory 2: Roche site). The interlaboratory measurement bias was −0.1% (95% CI interval from −0.7 to 0.5%) and the 2S interval of the relative difference was 6.5% (lower limit CI interval from −7.6 to −5.6%, upper limit CI interval from 5.4 to 7.4%).

Uncertainty of results

The estimation of uncertainty in the preparation of the calibrators was performed as a type B evaluation. All other aspects, e.g., calibration, sample preparation, and measurement and evaluation of the sample result, were evaluated as type A uncertainty in the precision experiment described above. Total measurement uncertainties and expanded measurement uncertainty for levetiracetam for single measurements were ≤2.0% and ≤4.0%, respectively, independent of the concentration level and the nature of the sample (Table 5) meeting the requirements defined in the introduction. The derived total uncertainty is multiplied by a coverage factor of k=2 to obtain an expanded uncertainty. The coverage factor corresponds to a confidence level of 95%, assuming a normal distribution.