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

Objectives: To describe and validate an isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure (RMP) for zonisamide to accurately measure serum and plasma concentrations. Methods: Quantitative nuclear magnetic resonance (qNMR) spectroscopywasemployedtodeterminetheabsolutecontent of the reference material used in order to establish trace-ability to SI units. Separation of zonisamide from known or unknown interferences was performed on a C8 column. For sample preparation a protocol based on protein precipitation in combination with a high dilution step was established. Assay validation and determination of measurement uncertainty were performed based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. Results: The RMP was proven to be highly selective and speci ﬁ c with no evidence of a matrix e ﬀ ect, allowing for quanti ﬁ cation of zonisamide within the range of 1.50 – 60.0 μ g/mL. Intermediate precision was <1.4 % and repeatability CV ranged from 0.7 to 1.2 % over all concentration levels. The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment ranged from 1.1 to 1.4 % and 0.8 – 1.0 %, respectively. Conclusions: We present a novel LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.


Introduction
The establishment of reference measurement procedures (RMPs) and the traceability of routine measurements to RMPs in accordance with the International Organization for Standardization (ISO) guidelines ISO 17511 [1] is considered a cornerstone of global comparability of analytical laboratory results.It provides an opportunity to establish consistent standards and best practices without bias contributions among in vitro diagnostic (IVD) manufacturers.Metrological control is provided by the Bureau International des Poids et Mesures (BIPM) and the National Metrology Institutes (NMIs).The IVD industry relies on reference materials and RMPs performed in reference or calibration laboratories to assign material values and to provide routine laboratories with traceable calibrator and control materials.If materials and/or methods are not provided by BIPM and NMIs, individual traceability concepts must be followed.Currently, in most cases only one or no calibration/reference laboratory is available for routine assignment of calibration values [2].
The generally accepted therapeutic range in epilepsy for zonisamide is between 10.0 and 40.0 μg/mL [10].Levels exceeding 40.0 μg/mL are associated with several side effects, such as weight loss, psychiatric effects (irritability, confusion, depression) and therapy may cause renal calculi [11].Moreover, sulfonamide, such as zonisamide, can cause severe allergic reactions, skin rashes, and blood disorders [12].Co-administration of zonisamide with other carbonic anhydrase inhibitors such as topiramate is not recommended in pediatric patients because of the risk of metabolic acidosis [13].The terminal elimination half-life of zonisamide is approximately 63 h [3].It is important to note that the half-life is affected by CYP3A4 inducers such as rifampicin or phenytoin [14].Therefore, TDM support is necessary when these drugs are co-medicated with zonisamide.
However, there is currently no RMP listed in the Joint Committee for Traceability and Laboratory Medicine (JCTLM) database [24] and thus standardization among laboratories is not guaranteed [2].Reference measurement systems are required to improve key parameters such as inter-laboratory measurement accuracy and inter-laboratory and inter-test variability [25,26].Such reference measurement systems consist of an RMP that must be virtually free of systematic errors (i.e., measurement bias) and must be linked to higher order reference materials.The measurement uncertainty of an RMP must be well below the allowable random error of the routine.As a rule of thumb, the goal is to be less than onethird of the routine error for sufficient error propagation reserve [27].
The reference materials for a particular analyte must be characterized with respect to their molecular identity and purity.Primary calibrators of defined concentrations can be prepared for the RMP to assign values to industrial master calibrators.From there, routine calibrators are used to determine the concentration in patient samples.To qualify as a "reference measurement laboratory" according to ISO 15195, the laboratory must implement RMPs of a higher order and the results produced must be accurate and traceable to national or international reference materials [28].
Quantitative NMR spectroscopy is a well-established primary method (primary ratio method) to determine the mass fraction (absolute content; g/g) of an analyte in a single, non-destructive experiment along with the potential of complete structure elucidation, when required using a plethora of 1D-and 2D-pulse sequences.Owing to powerful structure elucidation characteristics, the linear response to the amount of the analyte and direct traceability to the kilogram via the qNMR internal standards, this technique provides unparalleled ability for determination of the amount or 'counts' of a particular analyte.The highest order qNMR internal standards are traceable directly to NIST benzoic acid 350b (Coulometric) and/or NIST PS1 (Benzoic acid; first primary qNMR standard).Additionally, as per the latest IUPAC Technical Report, qNMR has been stated as a potential primary reference measurement procedure ideally suited for the characterization of primary reference materials (PRM) [29].
The objective of this study was to develop and validate an RMP for zonisamide that meets the requirements of the ISO 15193 guidelines [30].The analytical imprecision target can be defined either from the therapeutic range to be monitored or pharmacokinetic-based calculations.Following the approaches of Glick or Burnett as outlined by Steele and colleagues for several drugs in 2001 [31], routine TDM of zonisamide should not exceed a measurement uncertainty between 4.2 % (Burnett) and 7.5 % (Glick) with targets derived from the therapeutic range.Using Fraser's pharmacokinetic model, which considers the elimination half-life and dose interval of the drug, the imprecision should remain below 3.3 %.
A recent investigation into the inter-laboratory measurement uncertainty in proficiency testing showed that mass spectrometry-based did not always meet the goal with the observed uncertainty range of 7-15 % [32].The interlaboratory expansion factor can be corrected by applying the average inter-laboratory/intra-laboratory variance component factor of 0.8, published by Steele [33].This leads to an estimate of the routine intra-laboratory uncertainty range between 5.6 and 12.0 %.This is in good agreement with the measurement uncertainty data derived from the Burnett or Glick estimation model but clearly exceeds the threshold of 3.3 % set by the pharmacokinetic modelling approach.
Following the approaches of Braga and Panteghini, who conventionally recommend that higher order references and methods should be no more than one third of the measurement uncertainty of the routine method [27], the target measurement uncertainty for zonisamide should be less than 1.1 % if the pharmacokinetic Fraser model is applied and less than 1.9 % if the performance of current routine measurements is targeted.Naturally, it is essential that the bias of an RMP compared to the unknown and highly theoretical true measurement value any RMP is zero or as close to zero as possible [34].One possibility to minimize bias terms between RMPs is the highly transparent and unequivocal reference material characterization.Furthermore, only comparison studies, as realized in the RELA proficiency testing scheme, allow such estimations [35].For zonisamide such service is currently not in place.
Herein, we outline a novel candidate RMP for zonisamide that meets the requirements of the ISO 15193 guideline.To facilitate the reproduction of the candidate RMP by other laboratories, details are described in three supplementary documents focusing on the technical implementation of the test procedure, the qNMR-based reference material characterization, and the calculation of measurement uncertainty.

Materials and methods
A detailed operation procedure on the test procedure methodology can be found in the Supplementary Material 1.
Zonisamide qNMR for determination of the purity of the standard materials qNMR measurements were performed on a Jeol 600 MHz NMR with a He-cooled Cryoprobe.Single-Pulse-1 HNMR (Supplementary Figure 1) was utilized for the quantitation (Methylene protons, CH 2 SO 2 NH 2 , 2H) with an inter-scan delay of 70 s.These methylene protons are best suited as the quantifying signals since the 166 most susceptible functional group, to hydrolysis, -NH 2 functionalization, cyclization reactions, etc., is the -SO 2 NH 2 group and any chemical modification here would have direct influence on the chemical shift value of this singlet resonance.The qNMR internal standard, 1,3,5-trimethoxybenzene (Content: 99.96 ± 0.065 (k=1)), is a TraceCert CRM available from SigmaAldrich and as per the accompanying certificate of analysis (CoA), this material is directly traceable to NIST PS1 (primary qNMR standard).Additional details about NMR acquisition and FID processing parameters can be found in the Supplementary Material 2.

Preparation of calibrators and quality control (QC) samples
Two calibrator stock solutions were used to prepare eight matrix-based calibrators.Per stock solution, 60 mg of zonisamide was weighed in tin boats on a microbalance (XPR2, Mettler Toledo, Columbus, Ohio, USA).The substance was then dissolved in 5 mL DMSO using a volumetric flask to reach a concentration of 12.0 mg/mL.The concentrations of the stock solutions were calculated based on the purity of the reference material (99.4 ± 0.1 %, determined by qNMR) and the amount weighed.From these stock solutions, another two working solutions were prepared.
Two working solutions were prepared from these stock solutions.Eight spike solutions were prepared by diluting the stock and working solutions, which were then further used to prepare final matrix-based calibrator levels.Therefore, calibrator spike solutions were volumetrically diluted 1 + 99 (v/v) in native human serum matrix to produce matrix-based calibrators uniformly distributed from 1.50 to 60.0 μg/mL (7.07-283 μmol/L) (see Figure 1).
In addition, four matrix-based QC levels were prepared by weighing 50 mg zonisamide in tin boats.Final QC levels were then prepared in the same way as described for the calibrator levels.The concentrations of the QC levels were set at four critical control points: above the lower limit of quantification (2.60 μg/mL), below (8.00 μg/mL) and within (20.0 μg/mL) the therapeutic reference range, and at the laboratory alert level (40.0 μg/mL) [10] (see Figure 1).

ISTD solution
[ 2 H 4 , 15 N]-Zonisamide was dissolved in the appropriate amount of DMSO to give a 1 mg/mL stock solution, by volumetric addition of DMSO.The stock solution was stored at −20 °C until further use for a maximum of eight months.The ISTD working solution was prepared freshly on each day of sample preparation by a two-fold dilution of the stock solution: 60 µL DMSO was mixed with 40 µL ISTD stock solution followed by the addition of 3,900 µL Milli-Q-water to obtain a concentration of 10.0 μg/ mL.

Liquid chromatography mass spectrometry
An Agilent 1290 Infinity II LC system (Santa Clara, CA, USA), equipped with a binary pump, a vacuum degasser, an autosampler at 7 °C and a column compartment, was used for the chromatographic separation.Zonisamide was separated using an Agilent Zorbax Eclipse XDB-C8 column (100 × 3 mm, 3.5 µm, Santa Clara, CA, USA) which was maintained at 40 °C in the column compartment.The mobile phases consisted of 10 % methanol in Milli-Q-water (v/v) with 0.1 % acetic acid (A) and 95 % methanol in Milli-Q-water (v/v) (B).
All measurements were performed at a flow rate 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, switching the eluent flow until 0.8 min and from 6.5 min to the waste.
Zonisamide was detected by multiple reaction monitoring (MRM) using an AB Sciex Q-Trap 6500+ mass spectrometer (Framingham, Massachusetts, USA) with a Turbo V ion source operating in negative electrospray ionization mode (ESImode).An optimized ion spray voltage of −3500 V and a temperature of 600 °C were applied.Nitrogen gas was used as curtain gas, collision gas, ion gas source 1, and ion gas source 2 and was set at 35, 10, 70 and 60 psi, respectively.
Several analyte-specific mass transitions were tested and optimized for intensity and reproducibility, resulting in the following transitions: m/z 211.1-119.1 (quantifier) and m/z 211.1-147.1 (qualifier).Monitoring of the quantifier/qualifier ratio in native matrix samples compared to the quantifier/qualifier ratio of neat system suitability test (SST) samples allowed checking for interfering substances in matrix samples, which should not differ by more than 20 %.Table 1 shows an overview of the selected reaction transitions as well as the remaining compound-dependent MS settings.

System suitability test
An SST was performed prior to each analysis to verify the system sensitivity, chromatographic performance, and potential carry-over effects.For this purpose, two SST samples, SST sample 1 and SST sample 2, were prepared corresponding to the analyte concentration within the processed calibrator level 1 and 8, respectively.
To pass the SST, the signal-to-noise ratio of the quantifier transition had to be ≥200 for SST sample 1 and retention times for SST sample 1 and 2 had to be within 3.8 min ± 0.5 min.Moreover, carry-over was assessed by injecting SST sample 2 followed by two blank injections.The analyte peak area in the first blank had to be ≤20 % of the analyte peak area of SST sample 1 to pass.

Calibration structure of analytical series and data processing
At the beginning and the end of the analytical series, the calibrators were measured in increasing concentrations.Both calibration functions were used to generate the final calibration function by quadratic regression of the area ratios of the analyte and the ISTD (y) against the analyte concentration (x) resulting in the function, y=a 0 + a 1 x + a 2 x 2 .Data processing was performed using Analyst software (version 1.6.3 or higher).Analyte and ISTD peaks were integrated within a retention time window of 3.8 ± 0.5 min using a smoothing and peak splitting factor of three, a noise percentage of 90 %, and a base sub-window of 0.5 min.

Method validation
Assay validation and determination of measurement uncertainty were performed based on existing validation guidelines such as the Clinical & Laboratory Standards Institute's C62A Liquid Chromatography-Mass   [37] and the Guide to the expression of uncertainty in measurement (GUM) [38].
Selectivity: Zonisamide and [ 2 H 4 , 15 N]-zonisamide were spiked into analyte-free native human serum, surrogate serum, and native Liheparin plasma to evaluate the selectivity for the quantifier and qualifier transitions.In addition, the isotopically labeled ISTD was spiked into analyte-free matrices to determine potential amount of residual unlabeled analyte within the labeled ISTD.The amount of unlabeled analyte in the ISTD must not exceed 20 % of the amount at the lower limit of the measuring interval (LLMI), which corresponds to the concentration level of the lowest calibrator.In addition, eight calibrator levels were prepared sixfold in neat solution (mobile phase A), native human serum, surrogate serum, and Li-heparin plasma to evaluate specificity.Standard lines were compared in terms of slope (n=6) and coefficient of determination (n=6).The 95 % confidence interval (CI) of the slopes must overlap and the coefficient of determinations must be ≥0.99 to exclude a potential ME.Additionally, calibrator samples in surrogate matrix, native serum and native plasma were evaluated as controls by applying neat calibration as standard.The recoveries were reported as the percentage of recovery of the measured concentration relative to the nominal concentration.
A comparison of the absolute peak areas of the analyte and the ISTD was also performed.Therefore, analyte and ISTD were spiked into neat solution (mobile phase A), native human serum, surrogate serum, and Li-heparin plasma after protein precipitation for three levels (8.00, 20.0 and 40.0 μg/mL) spread over the working range.Ion enhancement or suppression was assessed by comparing the peak areas of the analyte and ISTD of the matrix samples with those of the neat samples.All samples were prepared in five replicates.A value >100 % indicates enhanced ionization and <100 % indicates suppressed ionization.The percentage deviation should not exceed ±10 %.
Linearity: To determine linearity, calibrator samples were prepared six times, two preparations on three days (n=6).In addition, two spiked serum calibrators with final concentrations of 1.20 and 72.0 μg/mL were prepared to extend the calibration range by 20 % at lower and higher concentrations.Correlation coefficients and residuals (n=6) were determined and had to be ≥0.999 and randomly distributed.
In addition, the linearity of the method was demonstrated by the recovery of nine serially diluted samples.Therefore, calibrator levels 1 and 8 were mixed as follows 9 + 1, 8 + 2, 7 + 3, 6 + 4, 5 + 5, 4 + 6, 3 + 7, 2 + 8 and 1 + 9 (v/v).Measurement results must show a linear dependence with a correlation coefficient ≥0.999.Recovery was expressed as the percentage recovery of the measured concentration relative to the nominal concentration of the samples.
Lower limit of the measuring interval (LLMI) and limit of detection (LOD): The precision and accuracy at the LLMI were determined by measuring spiked serum matrix samples in the expected concentration range of the quantitation limit.The quantitation limit matches the lowest calibrator level (1.50 μg/mL).Sample preparation was repeated five times.Recovery, bias and precision were determined and had to be within the range determined in the accuracy and precision experiment.
To estimate the LOD, the approach of Armbruster et al. was used.Therefore, the limit of blank was calculated using 10 independent matrix blank samples as follows: LOB=mean blank + 1.645(SD blank ).The LOD was then estimated using 10 replicates of calibrator level 1, which was used as the low concentration sample: LOD=LOB + 1.645(SD low concentration sample ) [39].
Precision and accuracy: A five-day validation experiment was performed to evaluate the precision and accuracy of the method.Total method variability was estimated using an ANOVA based variance component analysis including variability components such as betweeninjection variability, between-preparation variability, betweencalibration variability and between-day variability.On each day, four spiked native serum and native Li-heparin plasma samples covering the measurement range (2.60, 8.00, 20.0 and 40.0 μg/mL) and two native patient serum samples were prepared in triplicate for part A and B and injected twice (n=12, measurements per day and n=60 measurements per five days).Independent calibration curves were generated for each part and day and used for quantitative analysis.Since the measurements had to be carried out under different conditions, one employee was responsible for the sample preparation of each of the parts A and B, and two different column batches were used.Data were evaluated using Biowarp, an internal statistic program based on the VCA Roche Open-Source software package in R [40].
Accuracy was evaluated using the same four spiked native human serum and native Li-heparin plasma samples.To demonstrate that the method can be used for highly concentrated samples, dilution integrity was determined using two spiked serum samples with concentrations of 64.0 and 80.0 μg/mL (dilutions 1 and 2).All samples were prepared in triplicate for each part A and part B (n=6, sample preparation) on one day.Accuracy was determined as the percentage recovery of the measured concentration relative to the spiked concentration, while trueness was reported as the percentage recovery of the mean measured concentration relative to the spiked concentration.

Sample stability:
The stability of processed native serum samples was evaluated at 7 °C.Therefore, calibrator and QC levels were measured again after seven days of storage and were compared to freshly prepared spiked serum samples.Recoveries were calculated by comparing the measured value with freshly prepared samples.The stability of native serum calibrator and control material stored at −20 °C was evaluated after four weeks.The recoveries were calculated by comparing the measured value with freshly prepared samples.The total error (TE) was used as an acceptance criterion and was estimated to be ±3 %, based on the results of the precision and trueness experiment.Stability can be guaranteed for a measurement interval of 2-28 days for y − 1 day, and for a measurement interval of >4 weeks for y − 1 week.
Equivalence of results between independent laboratories: The agreement of the RMP between two independent laboratories (Laboratory 1: Dr. Risch Ostschweiz AG, Buchs SG; and Laboratory 2: Roche Diagnostics GmbH, Penzberg) was assessed by a method comparison Schierscher et al.: An ID-LC-MS/MS-based candidate RMP for the quantification of zonisamide study including 77 native patient samples, 10 native patient sample pools and 30 spiked serum samples.All samples were prepared once and were measured over three days.Additionally, a three-day precision experiment was performed at Laboratory 2 based on the experimental design described the section "Precision and accuracy".The candidate RMP was transferred to laboratory 2 and system setup was applied as described in the Supplementary Material 1 with some modifications: an ultramicrobalance XP6U/M (Mettler Toledo) and aluminum weighing boats were used for the preparation of stock solutions.
Uncertainty of measurements: Measurement uncertainty was determined according to the GUM [3838] and Taibon et al. [41] and considered the following steps: purity of the reference material, 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.The estimation of the uncertainty in the preparation of the calibrators was performed as a type B evaluation.All other aspects were evaluated as type A evaluation.The total measurement uncertainty of the whole approach was estimated as a combination of type A and type B uncertainties.

Traceability to SI units
Traceability to the SI unit of mass (kilogram), the most important parameter for a reference measurement method, has been established by the utilization of 1,3,5-trimethoxybezene as a qNMR internal standard which is directly traceable to NIST PS1 (primary qNMR standard) as per the accompanying certificate of analysis (CoA).Furthermore, traceability to the SI unit of amount of substance (mole) is also included owing to NIST PS1.Since Planck's constant is now the main parameter for defining kilogram and Avogadro's constant for mole, qNMR methodology is best suited to fulfill both the traceability chains.Additionally, as per the latest IUPAC Technical Report, qNMR has been stated as a potential primary reference measurement procedure ideally suited for the characterization of primary reference materials [29].Six individual experiments (Supplementary Material 2 Table 1 and Figure 2), involving six individual weightings of the analyte and 1,3,5-trimethoxybenzene (qNMR ISTD) yield a final content value of 99.4 ± 0.1 % (k=1).

Selectivity
The developed gradient combined with a reversed phase column (Agilent Zorbax Eclipse XDB-C8) separated zonisamide well from polar and apolar matrix components with a retention time of 3.8 min (Figure 2).Selectivity was determined by analyzing sample pools of analyte-free native human serum, surrogate serum and native Li-heparin plasma.No signals were observed within the expected retention time window (3.8 ± 0.5 min).Moreover, no residual zonisamide was observed in the ISTD ion traces.Furthermore, the samples measured in the method comparison study showed no interference within the retention time window of zonisamide.

Matrix effect/specificity
Matrix-dependent effects were avoided by a high dilution of the sample after protein precipitation.This was demonstrated by the post-column infusion experiment, as no change in the ionization field was observed at the expected retention time of zonisamide, independent of the matrix tested.
Furthermore, a comparison of slopes and coefficients of determination in different matrices was performed.Calibrations showed mean slopes (n=6, sample preparations) of 0.0545 (95 % CI 0.0542-0.0549)for the neat solution, 0.0546 (95 % CI 0.0543-0.0550)for the native serum, 0.0547 (95 % CI 0.0544-0.0550)for the surrogate serum and 0.0549 (95 % CI 0.0545-0.0554)for the native Li-heparin plasma.The CIs of the slopes overlap, indicating that they are not significantly different.Moreover, mean r 2 values were ≥0.99 independent of the matrix used for calibration.Both results confirm the absence of a ME.Furthermore, matrix-based samples, when calibrated against neat, showed no significant bias ranging from −0.9 to 1.3 % with CVs of less than 1.9 %.
In addition, MEs were evaluated based on the approach of Matuszewski et al. [42].The recoveries of the analyte peak areas ranged from 101 to 108 % and the ISTD peak areas ranged from 101 to 109 % (Table 2).The recoveries are slightly above 100 % indicating an ion enhancement.However, the area ratio recoveries were between 99 and 100 %.This confirms the compensating effect of the labeled ISTD.

Linearity
The linearity of the method was demonstrated by analyzing calibration curves (n=6, sample preparations) extended by ±20 % of the assay measuring range (1.20-72.0μg/mL).The residuals were randomly distributed in a quadratic regression model and were therefore selected for assay calibration.The correlation coefficients were 1.00 for all individual calibrations.Based on these results, samples 1-11 were evaluated and showed a linear dependency with a correlation coefficient of 1.00.The relative deviation ranged from 0.1 to 1.2 % and the CV was determined to be ≤0.9 %.

Lower limit of the measuring interval and limit of detection
The LLMI was determined using spiked samples at the lowest calibrator level.The relative bias showed a deviation of 0.3 % and the CV was 1.7 % at a concentration of 1.50 μg/mL.The LOD was estimated to be 0.317 μg/mL with an intensity corresponding to approximately one-fifth of the average peak height of calibrator 1.

Precision and accuracy
Precision, accuracy, and trueness were determined using spiked serum and plasma samples at four concentration levels (2.60, 8.00, 20.0 and 40.0 μg/mL).Each level was prepared in triplicate and injected twice by two different operators (n=12) on five different days (n=60).Prior to the sample preparation, two highly concentrated samples (64.0 and 80.0 μg/mL) were diluted with matrix to determine accuracy and trueness for these samples (n=6, two operators).
Precision was evaluated in a multi-day validation experiment.To assess the overall variability of the method, variability components such as variability between injections, between preparations, between calibrations, and between days were determined using an ANOVA-based variance component analysis.Intermediate precision was found to be less than 1.4 % independent of the matrix.The repeatability CV ranged from 0.7 to 1.2 % over all concentration levels (Table 3).The evaluation of trueness within this experimental design was evaluated by comparing the measured data to the calculated sample concentrations.Therefore, data from the first day were evaluated (n=6, two operators).The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels.Highly concentrated native serum levels showed a bias between 0.9 and 1.1 % (Table 4).

Stability
Processed samples were found to be stable for six days at 7 °C with recoveries ranging from 98 to 102 %.Additionally, the stability of spiked frozen serum calibrator and QC material stored at −20 °C was evaluated after 28 days.Therefore, freshly prepared calibrators and QC levels were used to evaluate the frozen samples.The recoveries ranged from 98 to 101 % compared with the original value and are within the TE.Consequently, samples can be stored at −20 °C for 27 days.

Equivalence of results between independent laboratories
The method comparison study containing 117 samples (77 native patient serum samples, 10 native patient sample pools Table : Matrix effect data of three different matrices compared to neat analyte solutions.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 /set  * , whereby set  corresponds to the respective matrix samples and set  to the neat samples.and 30 spiked serum samples) showed that two samples were below the lower limit of quantitation and five samples were above the calibration range.Dilution of these samples was not possible due to lack of additional sample volume.Moreover, one spiked sample was highlighted as outlier using the LORELIA (local reliability) outlier test [43].Therefore, these samples were excluded from the evaluation.Passing-Bablok regression analysis showed a good agreement between the two laboratories and resulted in a regression equation with a slope of 1.02 (95 % CI 1.02-1.03)and an intercept of 0.02 (95 % CI −0.06 to 0.08).Moreover, the Pearson correlation coefficient was ≥0.999 (Figure 3A).The Bland Altman analysis plot showed a data scatter which is independent of the analyte concentration with a mean bias of 2.6 % (95 % CI interval from 2.2 to 3.1 %) and a 2S agreement of 4.8 % (lower limit CI interval from −3.0 to −1.4 %, upper limit CI interval from 6.6 to 8.2 %) (Figure 3B).
The y-axis within the Bland Altman Plot shows the difference of laboratory 2-laboratory 1, scaled by their mean.Naturally, both measurements come along with a certain measurement uncertainty.Thus, the uncertainty of the calculated difference of both measurements is respectively larger.Based on the uncertainty from calibrator preparation of ≤0.86 % an allowable bias for the RMP B ref ≤1.72 % can be assumed.The acceptable CV of the difference between two laboratories can be estimated based on the exact mathematical derivation for the CV of the ratio (lab2 − lab1)/ 0.5*(lab1 + lab2).It can be approximated by CV(lab2 − lab1) =√(CV (lab2) 2 + CV (lab1) 2 ).The performance evaluation in the laboratory 2 showed an intermediate precision of ≤2.6 % which is slightly higher to the intermediate precision of laboratory 1 of ≤1.2 %.Thus, following the TE idea, a maximum allowed difference between the two laboratories can be calculated as   laboratories, we can expect that most of all differences of the measurements of the individual samples fall between ±9.1 %, which is in good agreement with the observed data.

Uncertainty of results
The total measurement uncertainties for single measurements of serum samples ranged from 1.1 to 1.4 %, and the total measurement uncertainties for target value assignment (n=6, three measurements on two days) ranged from 0.8 to 1.0 %, independent of concentration level and sample type (Tables 5 and 6).
Consequently, the expanded measurement uncertainties are between 2.2 and 2.9 % for single measurements and between 1.6 and 2.1 % for multiple measurements.The derived total uncertainty is multiplied by a coverage factor of k=2 to obtain an expanded uncertainty.

Discussion
The focus of this project was to establish a candidate RMP for the quantification of zonisamide in human serum and plasma.The development of the RMP followed in its main LC-MS parameters the considerations for routine assays to ensure its overall comparability with routine operations.Hence reversed phase gradient elution chromatography coupled to tandem mass spectrometry with electrospray ionization was chosen as LC-MS/MS instrument setting.Successful RMPs rely on well-characterized reference materials, clear and transparent reporting of calibrator preparation, sample handling, analytical setup, and measurement uncertainties.In order not to compromise the quality of the method presented in this communication when this method is transferred to another laboratory, care must be taken not to overlook or forget any key elements as presented in this communication.
Since no primary reference material is available for zonisamide, qNMR characterized materials were used for the assay calibration.Utilizing qNMR allowed the establishment of the unbroken traceability chain from the RMP to the SI units (kg and mole).The preparation of calibrator solutions was presented in a transparent way including the associated uncertainty terms.Concentrations of calibrator and control samples were carefully optimized to cover the measurement range sufficiently; the pipetting scheme including the choice of pipet volumes and number of pipetting steps was optimized to minimize uncertainty contributions.
To minimize the uncertainty contribution of the measurement procedure itself, a single analyte RMP was designed.Thereby it was ensured, that LC-MS/MS instrument settings depending on physicochemical target analyte properties as the mass spectrometer ion source parameters and the chromatographic mobile phase composition were tailored to the specific needs of the analyte.In addition, single analyte monitoring allowed to realize the maximal possible number of data points across the recorded chromatographic peaks.Optimization steps towards the presented sample preparation scheme included fluid handling, selection of optimal pipettes; protein precipitation with equilibration times and dilution into the linear range of the MS detectora point to be remembered if the method is transferred to an instrument with different absolute ion yields.
The presented method fulfils the requirements for an RMP for zonisamide in terms of sensitivity, selectivity, and reproducibility.Measurement uncertainty for single measurements was found to be between the target uncertainty defined by the pharmacokinetic Fraser model and the target uncertainty derived by the performance of current routine measurements and can be further reduced performing multiple measurements (n=6).The presented study is free of matrix effects as shown by calibration slope comparison, post column infusion and ion yield attenuation experiments.
A second independent laboratory established the candidate RMP with no significant increase in interlaboratory-bias.It can be concluded that the protocols for the preparation of calibration solutions and samples are robust.The inter-laboratory comparison study also demonstrated that the method is effective in processing a large number of samples efficiently.This gives the operator confidence in this RMP for the evaluation of routine samples with unclear results.As a result, the method satisfies the demands to play a leading role in the traceability chain, and to perform method comparison studies and check problematic routine samples.

Conclusions
In this paper a LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma is presented.It may serve as traceable and reliable platform for the standardization of zonisamide routine assays and evaluation of clinically relevant samples.on behalf of Roche Diagnostics GmbH.Roche employees

Figure 1 :
Figure 1: Schematic overview of zonisamide calibrator and control levels chosen to allow optimal coverage of measurement and therapeutic reference range.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: 4.7.

Figure 2 :
Figure 2: Zonisamide LC-MS/MS derived analytical readouts.(A) Chromatogram of a matrix blank showing the analyte SRM ion trace (left) and ISTD SRM ion trace (right); (B) chromatogram of calibrator level 1 with a concentration of 1.50 μg/mL spiked in native serum (left) and ISTD (right); (C) pooled patient sample with a concentration of 5.65 μg/mL (left) and ISTD (right).
2 * B ref + 1.96 * √(CV (lab 2) 2 + CV (lab 1) 2 ), leading to 9.1 %.This means that based on the uncertainty calculation of the calibrator preparation and the precision of the two

Figure 3 :
Figure 3: Results from the patient sample-based zonisamide 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=109) samples between the independent laboratories (Laboratory 1: Risch site, and Laboratory 2: Roche site).The regression analysis resulted in a regression equation with a slope of 1.02 (95 % CI 1.02-1.03)and an intercept of 0.02 (95 % CI −0.06 to 0.08).The Pearson correlation value was ≥0.999.(B) Bland-Altman plot for the method comparison study of the RMP (n=109 samples) between two independent laboratories (Laboratory 1: Risch site, and Laboratory 2: Roche site).The inter-laboratory measurement bias was 2.6 % (95 % CI interval from 2.2 to 3.1 %) and the 2S interval of the relative difference was 4.8 % (lower limit CI interval from −3.0 to −1.4 %, upper limit CI interval from 6.6 to 8.2 %).

Table  :
Precision performance parameters for zonisamide quantification using the candidate RMP (n= measurements).
RMP, reference measurement procedure.Conversion factor µg/mL to µmol/L: ..The coefficients of variation for repeatability and intermediate precision, which were determined from the individual variances, are printed in bold.

Table  :
Bias and  % CI of native serum and native Li-heparin plasma samples (n=).The mean bias and corresponding confidence intervals were calculated using the individual sample biases of n= preparations.

Table  :
Overview of measurement uncertainty for zonisamide quantification with the candidate RMP in serum samples for single measurements.Patient sample  Patient sample  . μg/mL . μg/mL . μg/mL . μg/mL . μg/mL . μg/mL CV, coefficient of variation.Conversion factor µg/mL to µmol/L: ..The total 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  :
Overview of measurement uncertainty for zonisamide target value assignment (n=) with the candidate RMP in serum samples.
CV, coefficient of variation.Conversion factor µg/mL to µmol/L: ..The total measurement uncertainty of the whole approach for 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.