CalibrationARK Topiramate assay was calibrated using a full calibration (6-point) procedure. To perform a full calibration, the ARK Topiramate Calibrators (0.0, 2.0, 4.0, 8.0, 24.0, and 60.0 μg/mL) were assayed in duplicate. Calibration curve was veri� ed with controls. A typical calibration curve is shown in Figure below.

TM

Topiramate Assay on The Ortho-Clinical Diagnostics Vitros® 5,1 FS Chemistry SystemE. Hardy1, J. Valdez2

1 St. Mary’s Hospital Laboratory, Madison WI, 2 ARK Diagnostics, Inc., Sunnyvale CA

IntroductionABSTRACTBackgroundTopiramate (2,3:4,5-Di-O-isopropylidene-β-D-fructopyranose sulfamate, Topamax®) is used as adjunctive therapy or monotherapy for patients with partial onset or primary generalized tonic-clonic seizures as well as Lennox-Gastaut syndrome. Topiramate is also indicated for the prophylaxis of migraines. Recently, the International League Against Epilepsy issued updated guidelines for the TDM of AEDs (P.N. Patsalos et. al, 2008. Epilepsia, 49(7):1239–1276). Situations in which AED measurements are most likely to be of bene� t include (1) to establish a clinical benchmark for individual therapeutic concentration which can be used at subsequent times to assess potential causes for a change in drug response; (2) to aid diagnosis of clinical toxicity; (3) to assess compliance; (4) to guide dosage adjustment in patients when pharmacokinetic variability exists (e.g., children, the elderly, patients with associated diseases, drug formulation changes, drug-drug interaction, pregnancy, or dose-dependent pharmacokinetics).

ObjectiveTo evaluate the performance of the new ARK™ Topiramate Assay on the Ortho-Clinical Diagnostics VITROS® 5,1 FS Chemistry System.

MethodsThe ARK Topiramate Assay is a homogenous enzyme immunoassay for quantifying topiramate in human serum or plasma. The assay was evaluated on the VITROS® 5,1 FS using an open channel for application of user-de� ned method. The assay was calibrated using a six point calibration curve (0 to 60 µg/mL) where increasing reaction rate correlates to increasing topiramate concentration. Performance of the assay assessed precision, limit of quantitation, linearity, recovery, endogenous interference, speci� city and method comparison to FPIA.

ResultsPrecision of tri-level controls was 2.4% CV (2.5 µg/mL), 3.4% CV (9.8 µg/mL), and 1.9% CV (37.0 µg/mL). Limit of Quantitation was 1.5 µg/mL. The assay was linear from 1.2 to 60.0 µg/mL. Analytical recovery was within 10% for nominal values 1.5 to 45.0 µg/mL. Endogenous substances (lipids, bilirubin, hemoglobin and albumin) and other AEDs did not interfere with measurement of topiramate at the levels tested. Passing Bablok regression analysis for method comparison between ARK and FPIA resulted in a correlation of 0.97 and slope of 1.09 (n=63).

ConclusionsAnalytical utility of the ARK Topiramate Assay for quantifying topiramate in serum and plasma was demonstrated on the Ortho-Clinical Diagnostics VITROS® 5,1 FS Chemistry System.

Precision Precision was determined by assaying twenty replicates of each tri-level control containing topiramate. Means, percent recovery from target, standard deviation (SD), and coe� cients of variation (% CVs) were calculated for each level. The precision ranged from 1.9 to 3.4%.

Lower Limit of QuantitationLimit of quantitation was evaluated using pooled human serum supplemented with known amounts of topiramate and assayed 5 times. The LLOQ of the ARK Topiramate Assay is de� ned as the lowest concentration for which acceptable inter-assay precision (≤20% CV) and recovery (±15%) is observed. The criteria of LLOQ were met at 1.5 µg/mL; the precision was 4.0% CV and the recovery was 90.7%.

Analytical RecoveryAnalytical recovery was performed by adding concentrated topiramate drug (USP) into human serum negative for topiramate. Test sample concentrations were 1.5, 2.5, 5.0, 10.0, 15.0, 30.0, and 45.0 µg/mL. Five replicates of each sample were assayed. The results were averaged and compared to the theoretical target concentration and the percentage recovery was calculated. The amount of topiramate recovered from nominal ranged from 90.7% to 106.0%.

Speci� cityA high concentration of each compound was spiked into normal human serum with known level of topiramate (20.0 μg/mL) and assayed along with a serum control of topiramate. Measurement of topiramate resulted in ≤10% error in the presence of drug compounds at the levels tested. The following compounds did not interfere with the measurement of topiramate at the levels tested.

Endogenous InterferenceInterference studies were performed ay clinically high concentrations of the following potentially interfering substances in serum with known levels of topiramate (approximately 20.0 μg/mL). Each sample was assayed using the ARK Topiramate Assay, along with a serum control of topiramate. Measurement of topiramate resulted in ≤10% error in the presence of interfering substances at the concentrations tested.

LinearityLinearity studies were performed as suggested in CLSI/NCCLS Protocol EP6-A. Negative pooled human serum was supplemented with topiramate to give 60.0 µg/mL and then diluted proportionally. Nominal % Recovery of mean values versus expected values ranged 80.2 to 110.3%. Linearity at speci� c dilutions was considered acceptable if the percent di� erence was ±10% between the predicted 1st and 2nd order regressed values or ±15% ≤ 1.5 μg/mL.

Method ComparisonSixty-three (63) clinical specimens from patients treated with topiramate were analyzed usingthe ARK Topiramate Assay on the VITROS® 5,1 FS chemistry analyzer and by the Seradyn assay (FPIA). Topiramate values ranged 2.3 to 38.9 μg/mL by FPIA and 2.4 to 40.9 μg/mL by the ARK assay. Passing-Bablock regression was used to plot the comparison.

ConclusionsThe ARK Topiramate Assay is a homogeneous enzyme immunoassay intended for the quantitative determination of topiramate in human serum or plasma on automated clinical chemistry analyzers. The results obtained are used in the diagnosis and treatment of topiramate overdose and in monitoring levels of topiramate to help ensure appropriate therapy. The ARK Topiramate reagents, calibrators and controls are provided in liquid form ready-to-use.

The ARK Topiramate Assay demonstrated acceptable performance on the VITROS® 5,1 FS chemistry analyzer. Performance of the assay showed good precision, accuracy, speci� city and linearity with excellent correlation to FPIA.

The range of the assay is 1.5 to 60.0 µg/mL on the VITROS® 5,1 FS chemistry analyzer, based on LLOQ and Linearity of measurement.

United Kingdom National External Quality Assessment Scheme Heath Controls (UK NEQAS:United Kingdom National External Quality Assessment Scheme, Cardi� Bioanalytical Services Ltd., 16 Mount Stuart Square, Cardi� CF10 5DP U.K.) were evaluated. The samples were tested duplicate. The mean and percentage recovery were calculated for each sample.

Mean( g/mL)

Target( g/mL)

Recovery(%)QC Sample SD CV

(%) N

LOWMIDHIGH

2.510.040.0

99.497.692.6

2.59.8

37.0

0.10.30.7

2.43.41.9

202020

Mean( g/mL)

Conc. Tested(µg/mL) SD CV

(%) Recovery

(%) N

0.51.01.5

0.230.811.36

0.010.010.05

3.71.34.0

45.681.290.7

555

Mean( g/mL)

Target(µg/mL) SD CV

(%) Recovery

(%)

1.52.55.0

10.015.030.045.0

1.42.55.3

10.415.929.044.2

0.050.070.030.550.230.530.82

4.02.80.55.31.51.81.9

90.7100.0105.2104.0106.096.698.3

Mean percent recovery: 100.1

Compound Conc. (µg/mL) Compound Conc.

(µg/mL)CarbamazepineEthosuximideFelbamateGapapentinOxcarbazepineLamotrigine Levetiracetam

Phenobarbital Phenytoin Primidone TiagabineValproic Acid Vigabatrin Zonisamide

10050050010050

100200

4050

100200200150200

SubstanceAlbuminBilirubin (conjugated)Bilirubin (unconjugated)CholesterolGamma-GlobulinHemoglobinTriglyceridesUric Acid

12 g/dL70 mg/dL70 mg/dL

269 mg/dL12 g/dL

1000 mg/dL1001 mg/dL

30 mg/dL

94.5102.898.1

108.294.097.1

108.295.9

SubstanceConcentration

TopiramatePercent Recovery

*Tested below LLOQ

ExpectedValue Results 1st Order

predictedResults

PercentRecovery

PercentDifference

1st OrderpredictedResults

1.21.83.03.64.86.0

12.024.036.048.054.060.0

0.961.613.063.705.096.62

13.0824.0537.2351.2855.6063.48

80.289.4

102.0102.8106.0110.3109.0100.2103.4106.8103.0105.8

1.261.832.973.554.695.83

11.5422.9734.3945.8151.5357.24

1.121.712.893.474.655.83

11.6823.2434.6445.8651.4156.91

-11.5-6.9-3.0-2.0-0.8-0.11.11.20.70.1

-0.2-0.6

*

TPM Linearity Graph 1st Order

y = 0.952x + 0.1189R2 = 0.999

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0

Expected Values (µg/mL)

Obs

erve

d V

alue

s (µg

/mL)

TPM Linearity Graph 2nd Order

y = -0.0006x2 + 0.9856x - 0.0658R2 = 0.999

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0

Expected Values (µg/mL)

Obs

erve

d V

alue

s (µg

/mL)

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0

FPIA (µg/mL)

AR

K T

PM A

ssay

on

VIT

RO

S® 5

,1 F

S (µ

g/m

L) Sample ID Spiked Mean

(µg/mL)Consensus Mean

(µg/mL)ARK Vitros Mean

(µg/mL)Recovery

(% Spiked) Recovery

(% Consensus)AED 0308AED 0408AED 0508AED 0608AED 0808

7.61.9

11.337.75.7

7.41.9

11.238.15.7

7.31.5

10.734.25.8

96.178.994.790.7

101.8

98.678.995.589.8

101.8

250

300

350

400

450

500

0.0 10.0 20.0 30.0 40.0 50.0 60.0

Topiramate concentration (µg/mL)

Cal

ibra

tor

rate

(mA

bs/m

in)

B) Presence of drug

inhibited

H

G6PDH

H

A) Absence of drug

G6PDHactive

G6PDHactive

DRUGDRUG

H H

(absorbs at 340 nm)NAD+ NADH

G6PDHactive

ARK Topiramate Assay vs FPIA

SlopeIntercept (µg/mL)Corr Coef (r²)N

1.09- 0.36

0.9763

Two specimens >32.0 µg/mL were diluted and tested by FPIA.