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

  • Tobias Schierscher , Linda Salzmann , Neeraj Singh , Martina Bachmann , Anja Kobel , Janik Wild , Friederike Bauland , Andrea Geistanger , Lorenz Risch ORCID logo , Christian Geletneky , Christoph Seger and Judith Taibon EMAIL logo
Published/Copyright: February 27, 2024
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 62 Issue 7

Abstract

Objectives

Phenobarbital serves as an antiepileptic drug (AED) and finds application in the treatment of epilepsy either as monotherapy or adjunctive therapy. This drug exhibits various pharmacodynamic properties that account for its beneficial effects as well as potential side effects. Accurate measurement of its concentration is critical for optimizing AED therapy through appropriate dose adjustments. Therefore, our objective was to develop and validate a new reference measurement procedure (RMP) for the accurate quantification of phenobarbital levels in human serum and plasma.

Methods

A sample preparation protocol based on protein precipitation followed by a high dilution step was established in combination with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a C8 column to separate target analytes from known and unknown interferences. Assay validation and determination of measurement uncertainty were performed based on current guidelines. Selectivity and Specificity were assessed using spiked serum and plasma samples; to investigate possible matrix effects (MEs) a post-column infusion experiment and a comparison of standard line slopes was performed. Precision and accuracy were determined within a multiday precision experiment.

Results

The RMP was shown to be highly selective and specific, with no evidence of matrix interferences. It can be used to quantify phenobarbital in the range of 1.92 to 72.0 μg/mL. Intermediate precision was less than 3.2 %, and repeatability coefficient of variation (CV) ranged from 1.3 to 2.0 % across all concentration levels. The relative mean bias ranged from −3.0 to −0.7 % for native serum levels, and from −2.8 to 0.8 % for Li-heparin plasma levels. The measurement uncertainties (k=1) for single measurements and target value assignment were 1.9 to 3.3 % and 0.9 to 1.6 %, respectively.

Conclusions

A novel LC-MS/MS-based candidate RMP for the quantification of phenobarbital in human serum and plasma is presented which can be used for the standardization of routine assays and the evaluation of clinically relevant samples.

Keywords: phenobarbital; isotope dilution-liquid chromatography-tandem mass spectrometry; reference measurement procedure; SI units; traceability

Introduction

Barbiturates are a class of depressant drugs that derived from barbituric acid. Despite the synthesis of approximately 2,500 derivatives, a subset of 50 has been marketed as anticonvulsants, anesthetics, and hypnotics [1]. Notably, most of these agents have been replaced by safer, non-addictive and non-controlled drugs. Nevertheless, phenobarbital (C12H12N2O3, molecular weight=232.24 Da) is still widely used. Especially in developing countries, it is the drug of choice to treat epilepsy since it is inexpensive, effective, and usually taken with good compliance [2], [3], [4]. Phenobarbital prevents seizures by facilitating γ-aminobutyric acid (GABA)-mediated inhibition through allosteric interaction with the GABAA receptors [5, 6]. Mean channel open time is prolonged without affecting open frequency or conductance [7]. Sedative properties of phenobarbital are due to its ability to directly activate GABAA receptors [8]. Phenobarbital is metabolized slowly, resulting in a half-life of 70 to 140 h and is inhibited by other drugs (e.g., phenytoin and valproic acid), resulting in higher plasma concentrations [9]. Approximately 25 % of phenobarbital is excreted unchanged in the urine [10]. The remainder is metabolized in the liver to p-hydroxyphenobarbital and 9-D-glucopyranosylphenobarbital, both of which are pharmacologically inactive [11, 12]. The therapeutic range is from 10.0 to 40.0 μg/mL and the toxic level is reached at 50.0 μg/mL [13] and can lead to respiratory and cardiovascular depression, and death [14, 15].

Immunoassays are widely used for (therapeutic drug monitoring) TDM of phenobarbital [16], although they may be non-specific and may react with metabolites, thus leading to confounding results in plasma [17]. In contrast, spectrometric techniques (e.g., LC-MS/MS) are highly specific and sensitive, and the methods are based on state of the art analytical principles. However, LC-MS/MS methods lack standardization with many individual laboratories developing ‘in-house’ methods [18], [19], [20], [21], [22], [23]. Therefore, RMPs play a crucial role in the standardization and harmonization of patient results. Currently, there is an RMP listed by the Joint Committee for Traceability in Laboratory Medicine (JCTLM) which was also used for target value assignment of the reference material “Antiepilepsy Drugs in Frozen Human Serum, SRM 900a” published by NIST [24], [25], [26]. Unfortunately, this material is no longer commercially available. Since the status of the NIST RMP was unclear and we were not aware of reference laboratories that have instituted the NIST RMP, it was decided to establish and validate a new candidate RMP according to the requirements of the ISO 15193 guideline [27]. In this regard, a commercially available SI-traceable reference material was utilized, the target value assignment of which was established by qNMR methodology as per the accompanying certificate of analysis.

Based on the desired phenobarbital routine measurement uncertainty (k=1), the desired target measurement uncertainty (k=1) for the RMP was estimated as previously described in our published RMPs [28], [29], [30], [31]. It ranged from 0.6 to 7.5 % depending on which estimation model – either based on the therapeutic range (Glick [32], Burnett [33]) or on the half lifetime of the drug (Fraser [34]) was used as described by Steele et al. [35]. Whereas the Glick and Burnett figures of merit are feasible, the measurement uncertainty demanded by the Fraser model seems unreasonable low. It is an effect of dosing phenobarbital in rather short intervals, although it has a very slow clearance from the circulation. This therapeutic approach leads to very shallow exposure troughs over the time leading to a very stable steady state drug exposure. Investigations into proficiency data by Steele and coworkers showed that the between laboratory variance of phenobarbital measurements is 8.4 % thus suggesting an actual (real life) measurement uncertainty of 6.7 % (k=1) when the average inter-laboratory/intra-laboratory variance component factor of 0.8 was also applied [35]. Pauwels critically questioned the model approach of Fraser by assessing the intra-individual variability of drug levels in routine. They corrected their data for the analytical variability (CVa=3.7 %) and could show from 20 measurement pairs, that in clinical TDM use, the intra-individual (CVi) phenobarbital drug level variability is 19.8 %. From that data they derived desirable routine performance criteria of 9.9 % imprecision (CVa) and 8.3 % bias summing up to a total error of analysis (TEA) allowance of 24.6 % [36]. If the total error (TE) calculation model based on biological variation is set to the “optimal” analytical performance specification goal [37], a CVa of 5.0 % and a bias of 4.1 % must not be exceeded to meet the associated TEA goal of 12.3 %. The found CVa matches well with – and is consequently confirmed by – the CVa data previously derived from the proficiency data based top-down analysis performed by Steele.

Since the goal of phenobarbital TDM is the avoidance of overexposure, e.g., due to drug–drug interactions it seems feasible to target the measurement uncertainty based on the therapeutic range considerations or on measurement requirements based on biological variability data derived from patients in medical care. The analytical goals derived from the Fraser model based on pure pharmacokinetic considerations must be critically discussed as not in match with the clinical needs.

Taken together, it can be assumed that a phenobarbital assay, to serve state of the art phenobarbital TDM as RMP, must perform at a CVa level of approximate 1.7 % or better. This is to meet the performance target criterion by Braga et al. stating that analytical uncertainties of less than one third of routine services should be achieved by an RMP [38].

We present here a novel LC-MS/MS-based candidate RMP for the quantification of phenobarbital in human serum and plasma. This manuscript does only cover phenobarbital, a RMP for primidone will be covered in a separate report. To facilitate the reproduction of the candidate RMP by other laboratories, details are described in two supplementary documents focusing on the technical implementation of the test procedure and the calculation of measurement uncertainty.

Materials and methods

A full description of the methods, materials and equipment used is provided in Supplementary Material 1.

Chemicals and reagents

LC-MS grade methanol (CAS 67-56-1) was purchased from Biosolve (Valkenswaard, The Netherlands). Acetic acid (CAS 64-19-7, Art. No. A6283-100ML), dimethyl sulfoxide (DMSO) (CAS 67-68-5, ACS reagent, ≥99.99 %), and primidone (CAS 125-3-7) were purchased from Sigma Aldrich (Taufkirchen, Germany). HPLC grade isopropanol (CAS 67-63-0, Art. No. 34863) was purchased from Riedel de Haën (Seelze, Germany). The reference material of phenobarbital (CAS 50-06-6, Art. No. MM0265.00-0250, Lot G985509) and its deuterated internal standard (ISTD), [2H5]-phenobarbital (CAS 73738-05-3, Art. No. LGCAMP0265.80-01, Lot No. 19154), were purchased from LGC Mikromol (Luckenwalde, Germany). Native human serum (Art. No. S1-LITER) was obtained from Merck (Darmstadt, Germany), drug-free human serum (multi-individual pooled; defined as surrogate serum, ID No. 12095432001) was obtained from Roche Diagnostics GmbH, native plasma matrix (Li-heparin, K2-EDTA and K3-EDTA) was obtained from anonymized patient samples and water was purified in-house 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.

Preparation of calibrators and quality control samples

Two individual calibrator stock solutions were prepared, which were used for the preparation of working and spike solutions. These solutions were than used for the preparation of the final matrix-based calibrator levels.

Per stock solution, 75.0 mg of phenobarbital were weighed in tin boats on a micro balance (XPR2, Mettler Toledo, Columbus, Ohio, USA) and dissolved in 5 mL DMSO using a volumetric flask to reach concentrations of 15.0 mg/mL. The exact concentration of the stock solutions was calculated based on the purity of the reference material (99.7 ± 0.4 %, based on the certificate) and the amount of weighed phenobarbital. These stock solutions were further diluted with DMSO to obtain working solutions with concentrations of 1.50 mg/mL. Both, stock and working solutions were then used to prepare eight calibrator spike solutions in DMSO. Final matrix-based calibrators, uniformly distributed from 1.92 to 72.0 μg/mL (8.27 to 310 μmol/L) (see Figure 1), were prepared by a 1+99 dilution (v/v) into human serum matrix.

Figure 1: Schematic overview of set calibrator and control levels, which were chosen to allow an optimal coverage of measurement and therapeutic reference ranges. Black circles indicate calibrator spike solution, black triangles are QC spike solutions 1–4, the solid black line is the measurement range, the dashed black line is the therapeutic reference range, and the black diamond is the alert level. QC, quality control. Conversion factor µg/mL to µmol/L: 4.3.
Figure 1:

Schematic overview of set calibrator and control levels, which were chosen to allow an optimal coverage of measurement and therapeutic reference ranges. Black circles indicate calibrator spike solution, black triangles are QC spike solutions 1–4, the solid black line is the measurement range, the dashed black line is the therapeutic reference range, and the black diamond is the alert level. QC, quality control. Conversion factor µg/mL to µmol/L: 4.3.

Matrix-based quality control (QC) samples were prepared similarly to the calibrators using a third independent stock solution with a concentration of 20.0 mg/mL. The concentrations for the control levels were set at four critical control points: above the limit of quantification (2.60 μg/mL), below (8.00 μg/mL) and within (25.0 μg/mL) the therapeutic reference range, and at the laboratory alert level (50.0 μg/mL) (Figure 1).

ISTD solution

To prepare the ISTD stock solution, 900 µL [2H5]-phenobarbital dissolved in methanol (1.00 mg/mL) was mixed with 8,100 µL of DMSO (v/v) to obtain a 100 μg/mL ISTD stock solution, which was stored at −20 °C for up to 8 months. The ISTD working solution was freshly prepared on each day of sample preparation by diluting the stock solution. Therefore, 100 µL of ISTD stock solution was added to 3,900 µL Milli-Q water to reach a final concentration of 2.50 μg/mL.

Sample preparation

The following sample matrices can be used: native human serum, surrogate serum, and plasma (Li-heparin plasma, K2-EDTA, and K3-EDTA). Fifty microliter of sample specimen (native sample/calibrator/QC) was mixed with 100 µL of ISTD working solution in a 2 mL tube (Eppendorf Safe-Lock Tube, Eppendorf, Hamburg, Germany). Then, 1,000 µL precipitation solution (75% methanol in Milli-Q water [v/v]) was added and the sample centrifuged. Subsequently, 10 µL of the supernatant was diluted with mobile phase A in two steps (step 1: 1+99 [v/v], step 2: 1+4 [v/v]).

Liquid chromatography mass spectrometry

Chromatographic separation was accomplished with an Agilent 1290 Infinity II LC system (Santa Clara, CA, USA) equipped with a binary pump, a vacuum degasser, an autosampler and a column compartment. Chromatographic separation of phenobarbital and the prodrug primidone was achieved on an Agilent Zorbax Eclipse XDB-C8 column (100 × 3 mm, 3.5 µm, Santa Clara, CA, USA) which was kept in the column compartment at 40 °C. The mobile phases consisted of water/methanol 90+10 (v/v) with 0.1 % acetic acid (A) and methanol/water 95+5 (v/v) (B). The LC was performed with a flowrate of 0.6 mL/min using a gradient over 10 min. The injection volume was set at 5 μL. Switching the eluent flow to the waste until 0.5 min and from 6.5 min, by a divert valve, reduced the contamination of the MS system.

Phenobarbital was detected in multiple reaction monitoring (MRM) mode using an AB Sciex Triple Quad 6500+ and Q-Trap 6500+ mass spectrometer (Framingham, Massachusetts, USA) with a Turbo V ion source in negative electrospray ionization mode (ESI – mode). The ion source was optimized with a spray voltage of −3,500 V and a temperature of 600 °C. Nitrogen gas was used as curtain gas, collision gas, ion gas source 1, and ion gas source 2 and were set at 35, 10, 60, and 50 psi, respectively. The collision cell entrance potential, collision exit potential, declustering potential and dwell time was set at −10 V, −15 V, −30 V and 50 ms, respectively, for all mass transitions.

The quantifier transition (m/z 231.1 to 42.1) serves as the basis of the quantification method. An additional specific mass transition (qualifier, m/z 231.1 to 188.1) was monitored to exclude interfering substances in native matrix samples. The quantifier/qualifier area ratios of native matrix samples and neat system suitability test (SST) samples were compared and should not differ by more than 20 %. Table 1 provides an overview of the SRM transitions and the remaining compound-dependent MS settings.

Table 1:

MS/MS parameters of phenobarbital and [2H5]-phenobarbital.

Analyte Precursor ion, m/z Product ion, m/z EP, V CE, V CXP, V
Phenobarbital Quantifier 231.1 42.1 −10 −45 −15
Qualifier 188.1 −10 −14 −15
[2H5]-phenobarbital Quantifier 236.2 42.1 −10 −45 −15
Qualifier 193.1 −10 −14 −15

  1. EP, entrance potential; CE, collision energy; CXP, collision exit potential; MS/MS, tandem mass spectrometry.

Testing for system suitability

Prior to each analysis, the SST is performed to determine the system’s sensitivity, chromatographic performance, and carry-over effects. Concentration levels of the SST sample 1 and SST sample 2 corresponded to the analyte concentration within the processed calibrator level 1 and 8, respectively. For passing the SST, the signal-to-noise ratio of the quantifier transition had to be ≥50 for SST sample 1 and both SST samples had to show a retention time within 4.6 ± 0.5 min. For the examination of potential carry-over effects, the high concentrated SST sample 2 was injected, followed by two blank injections. The analyte peak area observed in the first blank after the injection of the SST sample 2 had to be ≤20 % of the analyte peak area of calibrator 1. The purity criterion for the first sample SST was applied to all further blanks in the measurement campaigns.

Calibration, structure of analytical series and data processing

Calibration is performed using calibrators prepared as described in the section ‘Preparation of calibrators and quality control samplesʼ. To generate the final calibration function, both calibrations were measured in increasing order at the beginning and at the end of an analytical series. The final calibration function was determined by a linear regression of the area ratios of phenobarbital and its ISTD (y) vs. the corresponding phenobarbital concentration (x), resulting in the function, y=ax × b.

Data evaluation was performed using the Analyst software, Version 1.6.3, employing the Intelli Quant algorithm (ABSciex). Phenobarbital and its ISTD showed a retention time of 4.6 min and were integrated within a 30 s window. A smoothing and peak splitting factor of 3 were used for peak integration. The noise percent was set to 90 % with a base sub window of 0.8 min.

Method validation

Assay validation and determination of measurement uncertainty were performed according to the Clinical & Laboratory Standard Institute (CLSI) Guidelines C62A Liquid Chromatography-Mass Spectrometry Methods [39], the International Conference on Harmonization guidance document Harmonised Tripartite Guideline Validation of Analytical Procedures: Text and Methodology Q2 (R1) [40] and the Guide to the expression of uncertainty in measurement (GUM) [41]. Further details are published in Taibon et al. [42].

Selectivity

To evaluate the selectivity of the method, native human serum, surrogate serum, and Li-heparin plasma were spiked with phenobarbital, primidone, and the ISTD [2H5]-phenobarbital at a concentration of 9.00 μg/mL. Analyte-free matrices were additionally checked for possible interfering matrix signals for the analyte quantifier and qualifier transitions. The residual amount of unlabeled analyte in the ISTD was determined to demonstrate the suitability of the ISTD used. Therefore, analyte-free matrices were spiked with ISTD, and the amount of residual unlabeled analyte must not exceed 20 % of the lower limit of the measuring interval (LLMI), which corresponded to the concentration of the lowest calibrator level.

Matrix effect and specificity

To investigate possible matrix effects (MEs), a qualitative post column infusion experiment was performed. For this, a solution of 25 ng/mL phenobarbital in mobile phase A/mobile phase B 1+1 (v/v) was infused into the HPLC column effluent through a T-piece at a flow rate of 7 μL/min. Processed matrix samples (native human serum, surrogate serum, and native plasma [Li-heparin, K2-EDTA, and K3-EDTA]) were then injected into a stable analyte background signal and the change in the background signal was recorded. Any shift in the MRM analyte signal at the expected retention time would indicate a matrix component mediated effect on the ionization.

To further demonstrate the matrix independence of the method, the slopes of the standard lines (n=6 sample preparations) were compared in neat solution (mobile phase A), native human serum, drug-free human serum, and Li-heparin plasma [43]. The confidence intervals (CI) of the slopes must overlap and the coefficient of determination must be ≥0.99 to exclude a ME. Recovery of calibrator samples (n=6 sample preparations) in surrogate matrix, native human serum, and plasma was evaluated using neat calibrators as standards.

In addition, absolute areas of analyte and ISTD as well as area ratios were compared [44]. Therefore, analyte and ISTD solutions were spiked into native human serum, surrogate serum, Li-heparin plasma, and neat solution (75% methanol) after protein precipitation for three levels (8.00, 25.0, and 50.0 μg/mL) spread over the working range. To evaluate the ME, the mean peak areas of the analyte and ISTD were compared with those of the neat samples, not exceeding a percent deviation of ±10 %. All samples were prepared in five replicates (n=5 sample preparations).

Linearity

To determine the linearity, the calibrators were prepared twice a day for 3 days (n=6 sample preparations). Additionally, to extend the calibration range by ±20 % two serum samples were prepared at the lower and upper end of the measurement range (1.40 to 90.0 μg/mL, n=6 sample preparations). Residuals and coefficient of determination were determined and had to be randomly distributed and ≥0.999, respectively.

Furthermore, the linearity of the method was demonstrated based on the recovery of serially diluted samples using the preferred regression model for calculation. Using calibrator level 1 as sample 1 and calibrator level 8 as sample 11 nine additional samples were diluted as follows: 9+1 (v/v), 8+2 (v/v), 7+3 (v/v), 6+4 (v/v), 5+5 (v/v), 4+6 (v/v), 3+7 (v/v), 2+8 (v/v) and 1+9 (v/v). Measurement results must show a linear relationship with a correlation coefficient ≥0.999. Recovery will be reported as the percentage of recovery of the measured concentration relative to the nominal concentration of the sample pools.

Lower limit of the measuring interval and limit of detection

Accuracy, trueness, and precision of the LLMI were determined by measuring spiked native human serum samples whose concentration corresponded to the lowest calibrator level (1.92 μg/mL). Sample preparation was performed five times, and recovery, bias, and precision were determined.

To estimate the limit of detection (LOD), the approach of Armbruster et al. [45] was used. Therefore, the limit of blank was calculated using 10 independent matrix blank samples as follows: LOB=meanblank + 1.645(SDblank). The LOD was then estimated using 10 replicates of calibrator level 1, which was used as the low concentration sample: LOD=LOB + 1.645(SDlow concentration sample).

Precision and accuracy

To evaluate the precision and accuracy of the developed method, a 5 days validation experiment was performed as previously described [28], [29], [30], [31, 42, 46]. An ANOVA-based variance component analysis was used to evaluate the total method variability, which included variability components such as between-injection variability, between-preparation variability, between-calibration variability, and between-day variability.

In brief, four spiked native serum and native Li-heparin plasma samples (2.60, 8.00, 25.0 and 50.0 μg/mL) covering the measurement range and two native patient serum samples were prepared in triplicate and injected twice. This scheme was performed by two individuals in parallel, defined as validation part A and B, over 5 days (n=12 measurements per day, n=60 measurements per 5 days). For each part and day, independent calibration curves were generated and used for quantitative analysis. To further vary conditions, two batches of columns and two different sample weightings were used for these measurements. Biowarp, an internal statistic program based on the VCA Roche Open Source software package in R [47], was used to evaluate the data.

Accuracy was assessed using four spiked native human serum and Li-heparin plasma samples (2.60, 8.00, 25.0 and 50.0 μg/mL) as well as two spiked high concentrated serum samples (80.0 and 100 μg/mL; dilution 1 and 2). Three sample preparations were prepared for each part A and B on one day (n=6 sample preparations) and the percentage of recovery of the measured concentration relative to the spiked concentration determined.

Sample stability

The autosampler stability of processed samples (calibrators and QC levels) stored at 7 °C was evaluated by comparison to freshly prepared calibrators and QC levels after 7 days. The stability of matrix-based calibrators and controls stored at −20 °C was evaluated after 28 days. Therefore, frozen samples were compared to freshly prepared calibrators and QC levels. The total error (TE) was used as an acceptance criterion and estimated based on the results of the precision and accuracy experiment, resulting in a maximum TE of ±8 %. Stability can be ensured for a measurement interval of 2 to 28 days (x) for x−1 day, and for a measurement interval of >4 weeks (y) for y−1 week.

Equivalence of results between two independent laboratories

To assess the agreement of the candidate RMP between two independent laboratories (laboratory 1 Dr. Risch Ostschweiz AG, Buchs SG and laboratory 2 Roche Diagnostics GmbH, Penzberg) a method comparison study was performed using 127 samples (70 anonymized native serum samples, 27 native plasma samples, and 30 spiked samples) provided by laboratory 2. In addition, a 3 days precision experiment was carried out in laboratory 2 using the same spiked and native serum samples as in laboratory 1. The LC-MS system and laboratory equipment used were similar in both laboratories with the following modifications for site 2: an ultra-microbalance XP6U/M (Mettler Toledo) and aluminum weighing boats were used for the preparation of stock solutions. Calibrators were prepared independently in both laboratories as described in Supplementary Material 1.

Uncertainty of measurements

Measurement uncertainty was determined according to the GUM [41] and Taibon et al. [42], where the following parameters were considered: purity of the reference material based on the certificate, weighing of the analyte, preparation of stock, working, spike and calibrator solutions, preparation of the ISTD solution, sample preparation of the calibrators, measurement of the calibrators and generation of the calibration curve, preparation and measurement of the unknown samples as well as evaluation of the sample results. To assess the uncertainty in the preparation of the calibrators, a type B evaluation was performed, while all other aspects were evaluated as type A. The total measurement uncertainty was then estimated by combining the type A and type B uncertainties.

For the assignment of reference or target values, multiple sample preparations for each sample were performed on at least 2 different days and the result was calculated as the arithmetic mean (n=x). Further details can be found in Supplementary Material 2.

Results

Selectivity

The goal was to develop an assay with the highest possible selectivity, which was achieved by optimizing the gradient in combination with the use of a reversed phase column (Agilent Zorbax Eclipse XDB-C8). Thus, phenobarbital and primidone, which can be at least partially metabolized to phenobarbital (see Figure 2), were baseline separated within 10.0 min. The chromatographic resolution (R) was ≥4.1 for all matrices tested. Selectivity was further determined by analyzing sample pools of analyte-free native human serum, surrogate serum, and native Li-heparin plasma which showed no signals at the expected retention time. In addition, no other polar or apolar matrix components were detected as interferences in the retention time window of phenobarbital in the samples measured during the method comparison study. The ISTD used fulfilled the requirements and did not contain any unlabeled analyte.

Figure 2: Phenobarbital LC-MS/MS derived analytical readouts. Analyte on the left-hand side, phenobarbital ISTD on the right-hand side. (A) Chromatogram of primidone (grey) and phenobarbital (black) both with a concentration of each 9.00 μg/mL in native serum; (B) The lowest calibrator level peak (1.92 μg/mL) in native serum; (C) Patient pool (n>5, 9.40 μg/mL). ISTD, internal standard; LC-MS/MS, liquid chromatography-tandem mass spectrometry.
Figure 2:

Phenobarbital LC-MS/MS derived analytical readouts. Analyte on the left-hand side, phenobarbital ISTD on the right-hand side. (A) Chromatogram of primidone (grey) and phenobarbital (black) both with a concentration of each 9.00 μg/mL in native serum; (B) The lowest calibrator level peak (1.92 μg/mL) in native serum; (C) Patient pool (n>5, 9.40 μg/mL). ISTD, internal standard; LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Matrix effect and specificity

To reduce matrix-dependent effects caused by salts, proteins, and phospholipids, a protein precipitation protocol followed by a high dilution step was introduced. During the post-column infusion experiment, no significant shift in ionization to the expected retention time was observed in serum and plasma matrix (K2-EDTA plasma, K3-EDTA plasma, and Li-heparin plasma).

MEs were further investigated by comparing the slopes and coefficients of determination of calibrations in different matrices (native serum, surrogate serum, and native Li-heparin plasma) with the calibration in neat solution (mobile phase A). The mean slopes (n=6 sample preparations) were found to be 0.215 (95% CI 0.214 to 0.217) for neat solution, 0.215 (95% CI 0.214 to 0.216) for native serum, 0.218 (95% CI 0.216 to 0.220) for surrogate serum and 0.217 (95% CI 0.215 to 0.219) for native Li-heparin plasma. The CIs of the slopes overlap, indicating that they are not significantly different from each other and supporting the absence of MEs. Mean r2 values were ≥0.99 independent of the matrix used for calibration. When matrix samples were measured against the neat matrix, the relative bias for all matrices and levels ranged from −2.2 to 3.7 % with CVs of less than 4.5 %.

In addition, analyte peak areas, ISTD peak areas, and area ratios of spiked matrix samples were compared with spiked neat samples (75% methanol [v/v]) after protein precipitation. Recoveries of the analyte and ISTD areas were determined for all matrices and levels and ranged from 100 to 107 % and 100 to 105 %, respectively (Table 2). The corresponding area ratios were between 100 and 102 %, confirming the absence of a ME and compensating effect of the labeled ISTD. Overall, these data show that the ME of this method is negligible.

Table 2:

Matrix effect (ME) data of native serum, surrogate serum and plasma compared to neat analyte solution. Analyte peak areas, ISTD peak areas, and analyte/ISDT area ratios as used in analyte quantification were investigated. Means from five-fold analysis were used as data input. The relative matrix effect (ME) was calculated as ME (%)=set 2/set 1 × 100, where set 2 corresponds to the respective matrix samples and set 1 to the neat samples.

Phenobarbital level, conc. Analyte ISTD Ratio
Mean, % 95% CI, % Mean, % 95% CI, % Mean, % 95% CI, %
Level 1

8.00 μg/mL		 Native serum 101 98–105 101 98–104 100 99–101
Surrogate serum 100 98–102 101 97–104 100 98–101
Native plasma 102 101–104 103 100–105 100 99–101
Level 2

25.0 μg/mL		 Native serum 103 100–107 102 100–104 101 99–104
Surrogate serum 100 98–103 100 99–101 100 98–102
Native plasma 106 102–109 105 103–107 100 99–102
Level 3

50.0 μg/mL		 Native serum 104 98–110 103 100–107 101 98–104
Surrogate serum 105 100–110 103 100–107 101 99–104
Native plasma 107 101–113 105 102–109 102 100–104

  1. CI, confidence interval; ISTD, internal standard; ME, matrix effect.

Linearity

The linearity of the method was demonstrated by analyzing six native serum calibration curves covering ±20 % of the measurement range a (1.40 to 90.0 μg/mL). The residuals were randomly distributed in a linear regression model and were therefore selected for assay calibration. Coefficient of determination were 0.999 for all individual calibrations. Based on these results, the serially diluted samples 1 to 11 were evaluated and showed a linear relationship with a coefficient of determination of 0.999. The relative deviation ranged from −0.7 to 2.4 % and the CV was determined to be ≤2.7 %.

Lower limit of the measuring interval and limit of detection

The LLMI was defined using spiked samples at the lowest calibrator level (1.92 μg/mL). Relative bias showed a deviation of 2.3 % and the CV was found to be 0.5 %. The LOD was estimated to be 0.697 μg/mL.

Accuracy and precision

Accuracy and precision were evaluated within a multi-day validation experiment using four spiked serum and plasma samples (2.60, 8.00, 25.0 and 50.0 μg/mL) and two native patient samples. Two different operators prepared each level in triplicate on 1 day over 5 different days and the samples were injected two times (n=60, measurements). Dilution integrity of the method was demonstrated using high-concentrated samples (80.0 and 100 μg/mL).

Variability components as between injections, between preparations, between calibrations, and between days were determined using an ANOVA-based variance component analysis. Intermediate precision including variances as between-day and -calibration was found to be less than 3.2 %. The repeatability CV including between-preparation and -injection ranged from 1.3 to 2.0 % across all concentration levels and regardless of the type of sample (Table 3).

Table 3:

Precision performance parameters for phenobarbital quantification using the candidate RMP (n=60 measurements).

Variance source Serum samples CV, %
Level 1 Level 2 Level 3 Level 4 Patient sample 1 Patient sample 2
2.60 µg/mL 8.00 µg/mL 25.0 µg/mL 50.0 µg/mL 9.60 µg/mL 20.7 µg/mL
Intermediate precision 3.2 1.9 1.7 1.9 1.9 2.3
Between-day 0.0 0.8 0.0 0.0 0.7 0.0
Between-calibration 2.7 0.0 1.0 0.8 0.8 1.5
Repeatability 1.7 1.8 1.4 1.7 1.6 1.7
Between-preparation 0.4 0.9 0.6 0.7 0.9 0.0
Between-injection 1.6 1.5 1.2 1.5 1.4 1.7
Variance source Plasma samples CV, %
Level 1 Level 2 Level 3 Level 4
2.60 µg/mL 8.00 µg/mL 25.0 µg/mL 50.0 µg/mL
Intermediate precision 3.0 1.7 1.5 1.9
Between-day 0.0 0.0 0.0 0.0
Between-calibration 2.3 0.6 0.8 1.2
Repeatability 2.0 1.6 1.3 1.5
Between-preparation 0.0 0.6 0.3 0.0
Between-injection 2.0 1.5 1.2 1.5

  1. CV, coefficient of variation; RMP, reference measurement procedure; Conversion factor µg/mL to µmol/L: 4.3. The coefficients of variation for repeatability and intermediate precision, which were determined from the individual variances, are printed in bold.

The trueness was determined using spiked samples by comparing the mean measured and calculated concentrations. The relative mean bias (n=6 sample preparations) within the measurement interval ranged from −3.0 to −0.7 % for serum samples and from −2.8 to 0.8 % for plasma samples. High concentrated samples showed a bias between −4.1 and −1.1 % (Table 4).

Table 4:

Bias and 95% CI of native serum and native Li-heparin plasma samples (n=6). The mean bias and corresponding confidence intervals were calculated using the individual sample biases of n=6 preparations.

Phenobarbital level, conc. Native serum Native plasma
Mean bias, % 95% CI, % Mean bias, % 95% CI, %
Level 1 2.60 µg/mL −0.7 −4.7 to 3.3 0.8 −2.2 to 3.8
Level 2 8.00 µg/mL −0.9 −2.5 to 0.6 −2.8 −4.1 to −1.6
Level 3 25.0 µg/mL −3.0 −4.2 to −1.8 −2.8 −3.6 to −1.9
Level 4 50.0 µg/mL −1.4 −3.1 to 0.3 −2.5 −4.4 to −0.5
Dilution 1 80.0 µg/mL −1.1 −3.1 to 0.9 – –
Dilution 2 100 µg/mL −4.1 −6.7 to −1.5 – –

  1. CI, confidence interval. Conversion factor µg/mL to µmol/L: 4.3.

Stability

Samples were found to be stable at 7 °C for 6 days on the autosampler with recoveries ranging from 96 to 104 % when compared to freshly prepared serum samples. Stability of spiked frozen serum calibrator and QC materials stored at −20 °C was shown for 27 days. The recoveries ranged from 94 to 102 % compared to freshly prepared serum calibrators.

Equivalence of results between independent laboratories

The method comparison study containing 127 samples (native serum and plasma patient samples as well as spiked samples) was performed between site 1 (Dr. Risch Ostschweiz AG, Buchs SG) and site 2 (Roche Diagnostics GmbH, Penzberg). Four samples were found to be below the lower limit of measuring interval and therefore excluded from the study. In addition, three samples were highlighted as outliers using the LORELIA outlier test [48]. Two of the samples were most likely mixed up. Passing–Bablok regression analysis showed a good agreement between the two laboratories and resulted in a regression equation with a slope of 1.00 (95% CI 0.99 to 1.01) and an intercept of 0.15 (95% CI 0.00 to 0.27). The Pearson correlation coefficient was ≥0.998 (Figure 3A). The Bland–Altman analysis showed a mean bias of 1.0 % with a 95% CI interval from 0.4 to 1.6 (Figure 3B). The data scatter was independent of the analyte concentration, ranging from −5.6 to 7.6 % (lower limit CI interval from −6.6 to −4.6 %, upper limit CI interval from 6.5 to 8.6 %).

Figure 3: Results from the patient sample-based phenobarbital 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=120 patients) between the independent laboratories (site 1 Dr. Risch Ostschweiz AG and site 2 Roche Diagnostics GmbH). Passing–Bablok regression analysis resulted in a regression equation with a slope of 1.00 (95% CI 0.99 to 1.01) and an intercept of 0.15 (95% CI 0.00 to 0.27). The Pearson correlation value was ≥0.998. (B) Bland–Altman plot resulted in an inter-laboratory measurement bias of 1.0 % (95% CI interval from 0.4 to 1.6 %) and a 2S interval of the relative difference of 6.6 % (lower limit CI interval from −6.6 to −4.6 %, upper limit CI interval from 6.5 to 8.6 %).
Figure 3:

Results from the patient sample-based phenobarbital 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=120 patients) between the independent laboratories (site 1 Dr. Risch Ostschweiz AG and site 2 Roche Diagnostics GmbH). Passing–Bablok regression analysis resulted in a regression equation with a slope of 1.00 (95% CI 0.99 to 1.01) and an intercept of 0.15 (95% CI 0.00 to 0.27). The Pearson correlation value was ≥0.998. (B) Bland–Altman plot resulted in an inter-laboratory measurement bias of 1.0 % (95% CI interval from 0.4 to 1.6 %) and a 2S interval of the relative difference of 6.6 % (lower limit CI interval from −6.6 to −4.6 %, upper limit CI interval from 6.5 to 8.6 %).

Precision performance evaluation within the second laboratory showed CVs for intermediate precision and repeatability of ≤2.4 % and ≤1.8 %, respectively. The results showed that the suggested RMP is transferable between independent laboratories and meets the requirements.

Uncertainty of results

The estimation of uncertainty of the calibrators was done as a type B evaluation. In the precision experiment all other aspects, e.g., calibration, sample preparation, measurement, and evaluation of the sample result, were evaluated as type A uncertainty. Measurement uncertainties (k=1) for single measurements of serum samples ranged from 1.9 to 3.3 % and was reduced to 0.9 to 1.6 % for target value assignment (n=6, three measurements on 2 days) (Tables 5 and 6). With the repeated measurements scheme in target value assignment the CVa figure of merit derived from routine data (see Introduction) can be met [32, 36]. Consequently, the expanded measurement uncertainties (k=2) ranged from 3.8 to 6.6 % for single measurements and from 1.9 to 3.3 % for target value assignment.

Table 5:

Overview of measurement uncertainty for phenobarbital quantification with the candidate RMP in serum samples for single measurements.

Level
Level 1 Level 2 Level 3 Level 4 Patient sample 1 Patient sample 2
2.00 µg/mL 8.00 µg/mL 25.0 µg/mL 50.0 µg/mL 9.60 µg/mL 20.7 µg/mL
Type B uncertainty:

calibrator preparation, CV, % 		0.87 0.88 0.80 0.76 0.88 0.80
Characterization of reference material 0.2 0.2 0.2 0.2 0.2 0.2
Preparation of:
 Stock solution 0.31 0.31 0.31 0.31 0.31 0.31
 Working solution 0.49 – – – – –
 Spike solution 0.62 0.63 0.51 0.45 0.63 0.45
 Matrix-based calibrator 0.87 0.88 0.80 0.76 0.88 0.80
Type A uncertainty:

intermediate precision, CV, % 		3.2 1.9 1.7 1.9 1.9 2.3
Measurement uncertainty (k=1), CV, % 3.3 2.1 1.9 2.0 2.1 2.4
Expanded measurement uncertainty (k=2), CV, % 6.6 4.3 3.8 4.0 4.2 4.8

  1. CV, coefficient of variation; RMP, reference measurement procedure. Conversion factor µg/mL to µmol/L: 4.3. The measurement uncertainty of the whole approach for a single measurement estimated as a combination of the uncertainty of calibrator preparation (type B uncertainty) and uncertainty of the precision experiment (type A uncertainty) are given in bold.

Table 6:

Overview of measurement uncertainty for phenobarbital target value assignment (n=6) with the candidate RMP in serum samples.

Level
Level 1 Level 2 Level 3 Level 4 Patient sample 1 Patient sample
2.00 µg/mL 8.00 µg/mL 25.0 µg/mL 50.0 µg/mL 9.60 µg/mL 20.7 µg/mL
Type B uncertainty:

calibrator preparation, CV, % 		0.87 0.88 0.80 0.76 0.88 0.80
Characterization of reference material 0.2 0.2 0.2 0.2 0.2 0.2
Preparation of:
 Stock solution 0.31 0.31 0.31 0.31 0.31 0.31
 Working solution 0.49 – – – – –
 Spike solution 0.62 0.63 0.51 0.45 0.63 0.45
 Matrix-based calibrator 0.87 0.88 0.80 0.76 0.88 0.80
Type A uncertainty:

intermediate precision, CV, % 		1.4 0.9 0.5 0.7 0.7 1.0
Measurement uncertainty (k=1), CV, % 1.6 1.3 0.9 1.0 1.1 1.3
Expanded measurement uncertainty (k=2), CV, % 3.3 2.5 1.9 2.1 2.3 2.5

  1. CV, coefficient of variation. Conversion factor µg/mL to µmol/L: 4.3. The measurement uncertainty of the whole approach for a target value assignment estimated as a combination of the uncertainty of calibrator preparation (type B uncertainty) and uncertainty of the precision experiment (type A uncertainty) are given in bold.

Discussion

The presented RMP candidate based on ID-LC-MS/MS allows the measurement of phenobarbital levels in human serum and plasma. The design was developed based on routine measurements and targeted therapeutic analyte concentrations in human serum and plasma specimens. To prepare samples for the selected analytical method, tandem MS with ESI in the negative mode and reversed-phase chromatography, protein precipitation followed by dilution was found to be more effective than liquid–liquid extraction or solid-phase extraction.

The uncertainty contribution of the measurement was reduced by designing a single analyte RMP. This ensured that LC-MS/MS instrument settings, such as the MS source parameters and the LC mobile phase compositions, were tailored to the specific needs of phenobarbital based on its physicochemical target analyte properties. One may argue that an experimental limitation of the presented procedure might be, that the quantifier transition relies on the detection of a rather small fragment with a mass of 42 Da. This fragment (an amide moiety) however showed high, stable intensities and was not affected by interferences. Since it is also commonly used in research and routine practices, we envisioned that this fragment was a good fit for setting up a phenobarbital candidate RMP [23, 49], [50], [51], [52].

Optimization of the presented sample preparation included fluid handling, selection of optimal pipettes; protein precipitation with equilibration times and dilution into the linear range of the MS detector – a point to remember when the point is transferred to an instrument with different absolute ion yield. Designing the RMP as single analyte method allowed to maximize the number of data points across the recorded chromatography peaks, which lead to a better (less fluctuating) peak area determination by the integration software.

The validation study confirmed that the developed analytical method met the sensitivity, selectivity, and reproducibility requirements for an RMP. In addition, the absence of MEs was evaluated using a calibration slope comparison and an ion yield attenuation experiment. The measurement uncertainty for multiple measurements (n=6) was found to be within the desired target measurement uncertainty of 3.4 % (k=2).

The successful transfer of the method to a second independent laboratory demonstrated that such a transfer can be performed without significant inter-laboratory bias. This indicates that the calibration solution preparation and sample preparation protocols are robust. In addition, the method comparison study showed that the method is capable of processing a high volume of patient samples in a relatively short time, making it suitable for taking a leading role in the traceability chain, conducting method comparison studies and resolving problematic routine samples.

Although the JCTLM database stated that the phenobarbital reference measurement procedure is still present, the activity status of the NIST RMP was unclear. In addition, there is no measurement service listed [53]. Since we received no positive answer of NIST on the current availability of the JCTLM procedure C10RMP6, a formal comparison of this candidate RMP to the existing RMP was not possible. Due the lack of NIST based reference material (see introduction) not even an indirect comparison was feasible.

Another limitation of the performed method transfer might be that the instrumentation was rather similar in both laboratories. We are however very confident that changing equipment to other vendors will not influence the central figures of merit of the RMP outlined here. We did prove this in the past [54], that method transfer to other LC-MS/MS setups is unproblematic. However, we encourage the readership that if such undertaking is carried out, that central parameters as the LLMI, the measurement range, the phenobarbital retention time and the recorded SRM must be closely monitored to be comparable to the method presented here.

Conclusions

In this paper a modernized LC-MS/MS-based candidate RMP for phenobarbital in human serum and plasma is presented which serves as traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Corresponding author: Judith Taibon, PhD, Roche Diagnostics GmbH, Penzberg, Germany, Nonnenwald 2, 82377 Penzberg, Germany, Phone: +49 8856 6012941, E-mail:

Acknowledgments

We would like to thank Aline Hoffmeister, Monika Kriner, Alexandra Herbik, Marion Deuster and Michael Dedio for their support in selecting and providing samples as well as Carina Schäfer for her contribution in the laboratory.

  1. Research ethics: All procedures were in accordance with the Helsinki Declaration. All samples used were exclusively anonymized leftover samples.

  2. Informed consent: Not applicable.

  3. Author contributions: All authors confirm they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  4. Competing interests: Tobias Schierscher and Lorenz Risch are employees of Dr. Risch Ostschweiz AG. Linda Salzmann, Anja Kobel, Janik Wild and Christoph Seger were all employees of Dr. Risch Ostschweiz AG at the time the study was conducted. Judith Taibon, Martina Bachmann, Andrea Geistanger, and Christian Geletneky are all employees of Roche Diagnostics GmbH. Friederike Bauland is an employee of Chrestos Concept GmbH & Co. KG, (Girardetstraße 1-5, 45131 Essen, Germany) and did the work on behalf of Roche Diagnostics GmbH. Roche employees holding Roche non-voting equity securities (Genussscheine): Judith Taibon, Christian Geletneky, Andrea Geistanger. The authors state no conflict of interest.

  5. Research funding: None declared.

  6. Data availability: The raw data can be obtained on request from the corresponding author.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/cclm-2023-1104).

Received: 2023-10-09
Accepted: 2024-02-12
Published Online: 2024-02-27
Published in Print: 2024-06-25

© 2024 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/cclm-2023-1104
Keywords for this article
phenobarbital; isotope dilution-liquid chromatography-tandem mass spectrometry; reference measurement procedure; SI units; traceability
Creative Commons
BY 4.0

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. LC-MS/MS random access automation – a game changer for the 24/7 clinical laboratory
  4. Reviews
  5. Neurofilament light protein as a biomarker for spinal muscular atrophy: a review and reference ranges
  6. Differential diagnosis of ascites: etiologies, ascitic fluid analysis, diagnostic algorithm
  7. Opinion Papers
  8. Clinical Decision Support System in laboratory medicine
  9. Blood over-testing: impact, ethical issues and mitigating actions
  10. General Clinical Chemistry and Laboratory Medicine
  11. An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of zonisamide in human serum and plasma
  12. An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine in human serum and plasma
  13. An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of phenobarbital in human serum and plasma
  14. An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of primidone in human serum and plasma
  15. An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine-10,11-epoxide in human serum and plasma
  16. Should we depend on reference intervals from manufacturer package inserts? Comparing TSH and FT4 reference intervals from four manufacturers with results from modern indirect methods and the direct method
  17. Comparison of three chatbots as an assistant for problem-solving in clinical laboratory
  18. Evidence-based cutoffs for total and adjusted calcium: a major factor in detecting severe hypo- and hypercalcemia
  19. Minor head injury in anticoagulated patients: performance of biomarkers S100B, NSE, GFAP, UCH-L1 and Alinity TBI in the detection of intracranial injury. A prospective observational study
  20. A comparative evaluation of the analytical performances of premier resolution-high-performance liquid chromatography (PR-HPLC) with capillary zone electrophoresis (CZE) assays for the detection of hemoglobin variants and the quantitation of HbA0, A2, E, and F
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  23. Vitamin D and vitamin K status in postmenopausal women with normal and low bone mineral density
  24. Hematology and Coagulation
  25. An automatic analysis and quality assurance method for lymphocyte subset identification
  26. Cancer Diagnostics
  27. Machine learning-based delta check method for detecting misidentification errors in tumor marker tests
  28. Cardiovascular Diseases
  29. Analytical evaluation of the novel Mindray high sensitivity cardiac troponin I immunoassay on CL-1200i
  30. Infectious Diseases
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  32. Letters to the Editor
  33. Inadvertent omission of a specimen integrity comment – an overlooked post-analytical error
  34. Falsely elevated T3 due to interference of anti-T3 autoantibodies: a case report
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  36. Lessons learned from site-specific sampling and biological half-life of IGFII and IIE(68-88) peptide: a case study
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  38. Clinical pilot study on microfluidic automation of IGH-VJ library preparation for next generation sequencing
  39. Long-term effects of interventions applied to optimize the use of 25-OH vitamin D tests in primary health care
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