ARTICLE

Retrospective Analysis of Pediatric and Adult

Populations Using an LC-MS/MS Method for
Oxcarbazepine/Eslicarbazepine Metabolite
Grace M. Kroner,a Ronald L. Thomas,b and Kamisha L. Johnson-Davisa,b,*

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Background: Therapeutic drug monitoring of anti-epileptic drugs is important to manage seizure control in
patients with epilepsy. Oxcarbazepine is a second-generation anti-epileptic drug approved for use in pediatric
patients, and eslicarbazepine acetate is a newer generation drug used as adjunctive therapy and monotherapy for
partial-onset (focal) seizures. While several second and third generation anti-epileptic drugs have broader thera-
peutic efficacy in patients, these drugs can still have severe side effects and variable interpatient pharmacokinet-
ics. Consequently, there is a need for accurate and sensitive analytical methods to support therapeutic drug moni-
toring.
Methods: An assay improvement for a LC-MS/MS method was developed for the major metabolite of oxcarbaze-
13
pine and eslicarbazepine, licarbazepine (MHD), using a C-labeled form of the compound as the internal stan-
dard. Additionally, retrospective data analysis was used to compare the distribution of results observed in adult vs
pediatric patients.
Results: Accuracy and linearity across the analytical measuring range of 1 to 60 mg/mL was acceptable. Inter- and
intra-run precision was less than 6% at 3 concentrations tested. The limit of detection was determined to be
0.5 mg/mL. Significant interference from hemolysis, icterus, lipemia, or 187 other potential interferences was not
detected.
Conclusions: The improved assay for MHD was appropriate for clinical use. Retrospective data analysis showed
that pediatric and adult patients had a similar distribution of oxcarbazepine/eslicarbazepine metabolite concen-
trations in serum.

INTRODUCTION been approved for treating epilepsy since 1993

Epilepsy can manifest through different types of
seizures (either partial or generalized), and various
medications have been found to be more effective
with certain types of seizures. Several drugs have
(1). Unlike some of the first generation anti-
epileptic drugs (AEDs), these second and third
generation drugs usually have fewer drug–drug
interactions and less severe adverse effects. While
general therapeutic ranges in adult populations

a
Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT; bARUP Institute for Clinical and Experimental Pathology,
Salt Lake City, UT.
* Address correspondence to this author at: 500 Chipeta Way, Mail Code 115, Salt Lake City, UT 84108. Fax (801) 584-5207; e-mail kamisha.davis@
hsc.utah.edu.
Previous presentations: poster presentation at MSACL 2019 meeting in Palm Springs, CA.
Received May 26, 2020; accepted September 10, 2020.
DOI: 10.1093/jalm/jfaa179
C American Association for Clinical Chemistry 2020. All rights reserved.
V
For permissions, please email: journals.permissions@oup.com.

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ARTICLE MHD Method Validation and Retrospective Analysis

IMPACT STATEMENT
A mass spectrometry method was redeveloped and validated to improve analytical accuracy and specif-
icity for the quantification of the major metabolite of oxcarbazepine and eslicarbazepine. The improved LC-
MS/MS metabolite assay was appropriate for clinical use in both pediatric and adult populations and the
analytical gains in sensitivity and specificity will improve the accuracy of patient results.

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are available for many drugs, ranges specifically
for pediatric patients are not well established, de-
spite the fact that children comprise up to 25% of
all epilepsy cases (1).
Oxcarbazepine is a second generation AED tar-
geted for treatment of partial seizures (2) and esli-
carbazepine acetate was approved to treat
partial-onset seizures. Both drugs have structures
similar to carbamazepine, and both drugs are me-
tabolized to licarbazepine, monohydroxy deriva-
tive (MHD), which is the active form of the drugs,
and glucuronide metabolites for renal elimination.
MHD reduces seizures by inhibiting sodium chan-
nel function (2). Oxcarbazepine and MHD are 40%
bound by plasma proteins, while eslicarbazepine
is 30% bound (3). Side effects of these drugs in-
clude dizziness, fatigue, and vomiting (4, 5).
Published clinical studies have demonstrated
that liver or kidney impairments alter drug metab-
olism, and higher doses may be necessary in chil-
dren to reach an effective concentration in serum
(6). For example, one pharmacokinetic model
demonstrated that the weight-normalized clear-
ance was 0.083 L/hkg in a 15 kg, 96 cm child (ap-
proximately 3-4 years of age), and 0.060 L/hkg in a
35 kg, 135 cm child (approximately 11 years of
age), but only 0.046 L/hkg in a 70 kg, 176 cm adult
(7). In agreement with that model, MHD half-life
and mean specific area under the plasma
concentration-time curve (for 48 h) were both ap-
proximately 30% less in children 2-5 years of age
than in children 6-12 years of age (5). Given the
limited numbers of subjects available for these
studies, wider scale investigation of concentra-
tions observed in children relative to adults would
be helpful for clinical interpretation.
The goal for this study was to perform an assay
improvement for a liquid chromatography tandem
mass spectrometry (LC-MS/MS)-based assay for
the oxcarbazepine/eslicarbazepine metabolite.
The assay was modified to optimize the mass
transitions for MHD to improve specificity, imple-
ment a MHD-13C6 internal standard to enhance
assay precision, and change the analytical plat-
form to gain a higher dynamic range to advance
linearity, compared to the previous LC-MS/MS
method. After implementation of the improved
method, we performed a retrospective analysis of
patient results analyzed by the revised method to
compare concentrations from pediatric and adult
patient populations and to determine whether
our method had an analytical measurement range
that was suitable for the patient populations
tested.
MATERIALS AND METHODS
A mass-spectrometry based method was devel-
oped at our institution in order to monitor blood
concentrations of the major metabolite for oxcar-
bazepine and eslicarbazepine, MHD. A 13C-labeled
derivative MHD was used as an internal standard
in all of the samples (Cerilliant). Twenty-five

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MHD Method Validation and Retrospective Analysis
microliters of patient sample were mixed with 975
lL of internal standard (MHD-13C6)/precipitation so-
lution (50% methanol/50% acetonitrile) to precipi-
tate proteins in the sample, and the resulting
supernatant was mixed with 0.1% formic acid in wa-
ter (mobile phase A) before 5 mL was loaded onto
the instrument. Separation occurred on a
Phenomenex Kinetex C18 2.6 lm HPLC column (50
x 2.1 mm) with a switch to mobile phase B of 0.1%
formic acid in acetonitrile and a flow rate of 800 mL/
min at a column temperature of 35  C. The HPLC
run time was 2.5 min; the first 30 s was set at 10%
mobile phase B, followed by 90 s of 95% mobile
phase B, then finishing with 30 s of 10% mobile
phase B. The compounds were detected by multiple
reaction monitoring on an AB Sciex 4000 mass
spectrometer, using electrospray ionization in posi-
tive mode. The mass transitions used for the inter-
nal standard were 261.1 and 198.2 Da for the
quantitative peak and 261.1 and 185.2 Da for the
qualitative peak. For MHD, the mass transitions
used were 255.1 and 165.1 Da for the quantitative
peak and 255.1 and 179.1 Da for the qualitative
peak. A 5-point calibration curve was used for quan-
tification (using concentrations of 1, 5, 10, 30, and
60 mg/mL MHD (Cerilliant)). Ascent software (Indigo
BioAutomation) was used for peak area integration.
The previous method was performed on a
Waters TQD instrument in positive electrospray
ionization mode. The mobile phase was identical
to the optimized method; however, the column
utilized was a Waters Acquity UPLC HSS T3 1.8
mm, 2.1 x 50 mm and 2 mL was injected onto the
mass spectrometer. The AMR was 1–40 mg/mL
and the mass transitions used for the internal
standard, UCB 17025 (UCB Pharma), were 185.1
and 69.0 Da for the quantitative peak and 185.1
and 140.0 Da for the qualitative peak. For MHD,
the mass transitions used were 237.1 and
179.1 Da for the quantitative peak and 237.1 and
194.0 Da for the qualitative peak. This method had
imprecision (þ/-20% CV) and limited specificity
and quantitative accuracy.
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ARTICLE
Accuracy was evaluated by comparison of the
optimized method to the previous UPLC/mass-
spectrometry method, using 108 previously tested
samples (patient samples, CAP Proficiency Testing
samples, and spiked samples) over 5 days. Sample
concentration covered the analytical measurement
range (AMR) of the assay, 1 to 60 mg/mL. Linearity
was assessed by analyzing 5 calibrator samples
that span the AMR. Three replicates at each con-
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centration were measured on 5 different runs.
Recovery was tested by splitting 5 positive patient
samples into 2 aliquots each and spiking one of
them with 30 mg/dL of MHD. Both the fortified and
unfortified samples were tested in duplicate, and
from these results, the percent recovery was calcu-
lated. Intra-run imprecision was assessed by mea-
suring 20 replicates each of 3 levels of fortified
samples (low: 5 mg/mL, medium: 20 mg/mL, and
high: 50 mg/mL concentrations) in 1 day. Inter-run
imprecision was assessed by measuring 4 repli-
cates each of the 3 levels of fortified samples over
5 days. Fortified samples were prepared by adding
standard obtained from Cerilliant into blank
pooled plasma. The limit of quantification (LOQ)
was determined by testing at least 5 different con-
centrations below and above the expected limit of
quantification in triplicate on 5 different runs and
assigning the LOQ as the lowest concentration
measurable with CV less than 15%. The limit of de-
tection was established at the lowest concentra-
tion in which the analyte was present at the
correct retention time, with a signal to noise ratio
greater than 5, and imprecision within 20% CV.
Specificity was evaluated by fortifying a blank (neg-
ative) serum sample with 187 potentially interfer-
ing compounds and performing repeat testing to
assess for analytical interference. Additionally, 2
replicates each were analyzed after the addition of
hemolyzed, lipemic, or icteric material to evaluate
possible interferences. Finally, 32 negative samples
underwent post-column infusion with MHD to as-
sess ion suppression. EP Evaluator was used for
method evaluation analysis.
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ARTICLE MHD Method Validation and Retrospective Analysis

For the retrospective data analysis, we examined

2243 unique results for the oxcarbazepine or eslicar-
bazepine metabolite assay in serum/plasma at ARUP
Laboratories. We eliminated results from patients
with reported ages greater than 95 years (n ¼ 6).
Additionally, we eliminated results from patients with
more than one result; 175 results were eliminated.
Any results above or below the analytical measuring
range were eliminated from further analysis

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(n ¼ 149). Therapeutic reference intervals and toxic
concentrations were from the literature (6, 8, 9). All
statistical analyses were performed using R Studio
and Microsoft Excel. The protocol for collection of the
limited dataset was approved by the University of
Utah Institutional Review Board.

RESULTS

Accuracy Fig. 1. Method comparison between the newly

The accuracy of the improved LC-MS/MS
method on an AB Sciex 4000 instrument was
compared to the previous assay on a Waters TQD
instrument. One hundred and eight patient sam-
ples, previously analyzed on the Waters platform,
were re-extracted and analyzed on the AB Sciex
4000 platform (Fig. 1). The results from Deming re-
gression statistics were: slope: 0.834 6 0.023, y-in-
tercept: 2.2 6 0.7 (both shown as mean 6
standard deviation), R2 ¼ 0.96. The acceptance cri-
teria for accuracy was: slope: 0.8 6 15%, y-inter-
cept: 0-3, R2: 0.94 6 15%. The standard deviation
of residuals was 3.7. Of note, our slope criteria
were centered at 0.8, due to limitations of accu-
racy and specificity of the previous method, which
was demonstrated on CAP proficiency testing sur-
veys, as a positive bias trend.
Linearity
We determined the AMR of the assay to be 1 to
60 mg/mL. The slope was 1.029 and the y-intercept
was -0.057 with an observed error of 5.3% and R2
of 0.996. The standard deviation of the residuals
developed LC-MS/MS method and the UPLC MS
method. Dashed line is line of equivalence. Solid
line represents Deming regression.
was 1.45. Linearity across the AMR was within our
limits of 20% total allowable error and 10% allow-
able systematic error.
Recovery
The average recovery was 94%, meeting our
expectations of recovery between 85% and 115%.
Precision
Average intra-run imprecision was 5.2% and av-
erage inter-run imprecision was 4.3% (Table 1).
Sensitivity
The limit of quantification was 1 mg/mL and the
percent CV was less than 20%. The limit of detec-
tion was determined to be 0.5 mg/mL, where the
signal of the analyte appeared at the expected re-
tention time and exhibited a signal to noise ratio
greater than 5 (Fig. 2).

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MHD Method Validation and Retrospective Analysis ARTICLE

Table 1. Intra- and inter-run precision data.

Intra-Run Precision (n ¼ 20) Inter-Run Precision (n ¼ 20)

Mean Standard Deviation CV Mean Standard Deviation CV
Low 5.20 0.25 4.8% 5.35 0.29 5.4%
Medium 19.55 1.18 6.0% 20.47 0.76 3.7%
High 52.13 2.44 4.7% 47.83 1.77 3.7%
The units for the mean and standard deviation is mg/mL.

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Fig. 2. Chromatography of a sample at 0.5 mg/mL showed signal at the expected retention time (indi-
cated by arrow) and a signal to noise ratio greater than 5.

Specificity/Interferences compounds (Supplemental Fig. 1). The drug classes

No interference was detected after fortifying the for the compounds tested for interference were
negative sample with a cocktail containing 187 antidepressants, anticonvulsants, antipsychotics,

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ARTICLE MHD Method Validation and Retrospective Analysis

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Fig. 3. Frequency of results for A) pediatric patients and B) adult patients. Median concentration for pe-
diatric and adult patient populations is indicated by an orange or blue line, respectively. Therapeutic
intervals (3-35 mg/mL) are indicated by dashed black lines. Toxic concentration is indicated by a dashed
red line.

antidiabetics, benzodiazepines, barbiturates, significantly higher than the distribution of results

opioids, cardiac drugs, NSAIDS, stimulants, and il-
licit drugs. Additionally, accuracy for hemolyzed
(1800 mg/dL hemoglobin), lipemic (4700 mg/dL tri-
glycerides), or icteric (7.25 mg/dL bilirubin) samples
containing approximately 10 mg/mL MHD was
greater than 87%. Finally, ion suppression was not
observed in any of the 32 tested negative samples.
Retrospective Analysis
There were 1913 results from unique patients
used for retrospective data analysis. The pediatric
population was defined as patients less than
18 years of age in our patient population. There
were 531 pediatric patients (55% male), with an
age range from 0 to 17 years, and 1382 adult
patients (51% male), with an age range from 18 to
95 years. The distribution of results from pediatric
patients, with a median of 20 mg/mL, was
from adult patients, with a median of 17 mg/mL
(Wilcoxon test P-value ¼ <0.001, Fig. 3). However,
both populations had a similar proportion of
patients within the therapeutic range concentra-
tions: 92.0% of adult patients and 91.5% of pediat-
ric patients. There was a slightly higher proportion
of pediatric patients who had concentrations
above the 40 mg/mL threshold for toxicity (4.33%
of pediatric compared to 3.18% of adult patients).
Given the limited number of patients in the pedi-
atric group, we were unable to assess potential
variation across age within the pediatric group.
DISCUSSION
An LC-MS/MS assay to quantify MHD was rede-
veloped to provide accurate and precise

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642 JALM | 637–644 | 06:03 | May 2021
MHD Method Validation and Retrospective Analysis
therapeutic drug monitoring results to support
patient care. The previous method had limita-
tions with accuracy and specificity because the
method utilized a suboptimal product mass tran-
sition from the loss of a water molecule during
fragmentation. In addition, the method did not
incorporate an isotopically-labeled internal stan-
dard for MHD. These factors led to an unaccept-
able performance on a CAP ZE proficiency
testing survey. The proficiency test specimens
contained multiple antiepileptic drugs and our
reported results had a 30% positive bias in com-
parison to the mean, due to poor specificity. The
improved method employed mass transitions for
MHD that were optimal to minimize interfer-
ences caused by specimen matrix effects or
commonly prescribed drugs. In addition, use of
a MHD 13C-labeled internal standard improved
accuracy of quantification by coeluting with MHD
to compensate for ion suppression or enhance-
ment. These modifications, along with changing
the analytical platform, improved the specificity,
accuracy, and linearity in comparison to the pre-
vious assay. Performance of the new method on
a CAP ZE proficiency testing survey was accept-
able, within < þ/- 2.0 SDI.
Retrospective data analysis was used to evalu-
ate over 1900 pediatric and adult results. Our
results demonstrated that the distribution of
results was similar between pediatric and adult
patients and the median concentrations for
both populations were within the range of 3–
35 mg/mL. A slightly higher proportion of pediat-
ric patients had results above 40 mg/mL.
Elevated serum drug concentrations in pediatric
patients may be due to the use of higher doses
to provide equivalent MHD exposure to adult
patients. Previous research has documented
30% to 160% increased clearance in pediatric
patients compared to adults, leading to higher
dose requirements than adults to achieve simi-
lar MHD concentrations (10, 11). Shorter elimi-
nation half-lives in pediatric patients also
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May 2021 |
ARTICLE
contribute to the need for higher dosage or
more frequent dosing in some cases to avoid
dramatic fluctuations in drug concentrations (3).
During dosing optimization, these factors may
cause slightly higher concentrations to be ob-
served in pediatric patients. In addition, approxi-
mately 50% of patients were estimated to
require more than one AED to control their
seizures (1). Many AEDs have well-established
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drug–drug interactions and are known to affect
the pharmacokinetics of oxcarbazepine (3, 6).
Finally, adult patients may be more likely than
pediatric patients to have a stable dosing regi-
men; recommended doses for pediatric patients
depend on the patient’s weight, and so doses
may require more frequent adjustments as the
child grows.
Our retrospective data analysis was limited by
the lack of access to patient information such as
diagnosis, dosing regimen, and medication history
to determine if patients were prescribed either
oxcarbazepine or eslicarbazepine. Therefore, we
were unable to correlate serum/plasma drug con-
centration to drug dose. Another limitation of our
study was the lack of outcome data for patients to
determine whether seizures were controlled
when the drug concentrations were within the
therapeutic range or if patients displayed signs of
toxicity when serum MHD concentrations
exceeded the upper limit of the therapeutic range.
To mitigate these limitations, we removed results
from the data set for patients who had serial
measurements performed to optimize therapy.
Our data suggest that MHD concentrations in se-
rum were similar between adult and pediatric
populations.
In summary, we have developed and validated a
LC-MS/MS method for the quantification of MHD
to support therapeutic drug monitoring for oxcar-
bazepine and eslicarbazepine therapy. The assay
was validated according to CLSI guidelines for
mass spectrometry. The analytical method met cri-
teria for accuracy, imprecision, and specificity for
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ARTICLE MHD Method Validation and Retrospective Analysis

interferences from matrix effects due to icterus, SUPPLEMENTAL MATERIAL

lipemia, and hemolysis and commonly prescribed
drugs, and thus will provide accurate results to Supplemental material is available at The Journal
support patient care. of Applied Laboratory Medicine online.

Nonstandard Abbreviations: AEDs, anti-epileptic drugs; MHD, monohydroxy derivative; LC-MS/MS, liquid chromatography tan-
dem mass spectrometry; AMR, analytical measurement range; LOQ, limit of quantification.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the follow-

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ing 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 ac-
countable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are ap-
propriately investigated and resolved.

G.M. Kroner, statistical analysis; K.L. Johnson-Davis, administrative support.

Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclo-
sure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: R.L. Thomas, ARUP Laboratories.
Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research
Funding: None declared. Expert Testimony: None declared. Patents: None declared.

Role of Sponsor: No sponsor was declared.

Acknowledgments: The authors wish to thank Dave Davis for his assistance with data collection.

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