The authors have declared that no competing interests exist.
Conceived and designed the experiments: STK PCH. Performed the experiments: STK WBL HYSW YLSN PSW. Analyzed the data: STK WBL PKK. Contributed reagents/materials/analysis tools: PCH. Contributed to the writing of the manuscript: STK. Technical advice: SHL. Patients’ recruitment: SHL.
To facilitate therapeutic monitoring of antiepileptic drugs (AEDs) by healthcare professionals for patients with epilepsy (PWE), we applied a GC-MS assay to measure three AEDs: carbamazepine (CBZ), phenytoin (PHT) and valproic acid (VPA) levels concurrently in one dried blood spot (DBS), and validated the DBS-measured levels to their plasma levels. 169 PWE on either mono- or polytherapy of CBZ, PHT or/and VPA were included. One DBS, containing ∼15 µL of blood, was acquired for the simultaneous measurement of the drug levels using GC-MS. Simple Deming regressions were performed to correlate the DBS levels with the plasma levels determined by the conventional immunoturbimetric assay in clinical practice. Statistical analyses of the results were done using MedCalc Version 12.6.1.0 and SPSS 21. DBS concentrations (Cdbs) were well-correlated to the plasma concentrations (Cplasma): r = 0.8381, 0.9305 and 0.8531 for CBZ, PHT and VPA respectively, The conversion formulas from Cdbs to plasma concentrations were [0.89×CdbsCBZ+1.00]µg/mL, [1.11×CdbsPHT−1.00]µg/mL and [0.92×CdbsVPA+12.48]µg/mL respectively. Inclusion of the red blood cells (RBC)/plasma partition ratio (K) and the individual hematocrit levels in the estimation of the theoretical Cplasma from Cdbs of PHT and VPA further improved the identity between the observed and the estimated theoretical Cplasma. Bland-Altman plots indicated that the theoretical and observed Cplasma of PHT and VPA agreed well, and >93.0% of concentrations was within 95% CI (±2SD); and similar agreement (1∶1) was also found between the observed Cdbs and Cplasma of CBZ. As the Cplasma of CBZ, PHT and VPA can be accurately estimated from their Cdbs, DBS can therefore be used for drug monitoring in PWE on any of these AEDs.
Epilepsy is a neurological disease that requires chronic treatment with antiepileptic drugs (AEDs). To date, the most commonly used AEDs are still carbamazepine (CBZ), phenytoin (PHT) and valproic acid (VPA). These drugs have maximum efficacy and minimum toxicity when their plasma drug levels are within their therapeutic indexes. Hence, routine plasma concentration monitoring is recommended especially during dose adjustments, for compliance check and/or for adverse drug reaction investigation
Various biological matrices including cerebrospinal fluid, tear and saliva have been used for TDM
Earlier studies on concurrent monitoring of multiple AEDs from one DBS were done mostly with high performance liquid chromatography (HPLC) and included whole blood concentrations of AEDs such as carbamazepine, phenytoin, lamotrigine and barbiturates with limited clinical validation
In our population of PWE, CBZ, sodium valproate (VPA) and phenytoin (PHT) are the most popular antiepileptic drugs (AEDs) - used either as mono or polytherapy
This study has obtained approval from the SingHealth Institutional Review Board (CIRB No. 2011/269/A). Only PWE who had routine plasma CBZ, VPA and/or PHT, blood and liver biochemistry monitoring on the day of visit were approached for written consent prior to blood sampling.
Assuming constant analytical standard deviations, the sample size recommended for method validation was suggested to be at least 41 per AED based on the following information: range ratio = 2, α = 5%, power = 90%, standardized slope deviation of 4
Venous whole blood samples were collected in EDTA tubes. Two drops of blood from the withdrawn blood, ∼30 µL each, were spotted onto 903 cards (903 Neonate Blood Collection Cards, Whatman GmbH, Dassel, Germany) and dried at room temperature, 25°C for at least 3 hours. The rest of the whole blood was sent to hospital laboratory for plasma AED quantitations as per routine protocols. To maintain direct comparability with plasma levels, DBS samples were stored at −80°C until the day of analysis. The 3 AEDs were proven to be stable at −20°C and 25°C for at least 10 days at concentrations ranging from 0.5 mg/L to 100 mg/L (
The routine plasma AEDs quantifications were done in the hospital laboratory using particle enhanced turbidimetric inhibition immunoassays (Beckman Coulter Inc. Unicel DxC800, USA). The imprecisions in CV% (mean level, SD) for low, medium and high concentrations based on 21 data points over a period of typically 10 days are as following: i) Carbamazepine [range 2.0–20.0 mg/L] between run = 9.2% (4.2 mg/L, 0.39), 7.6% (10.79 mg/L, 0.82), 5.9% (15.24 mg/L, 0.93) and within run = 3.9% (3.92 mg/L, 0.15), 2.7% (10.41 mg/L, 0.28, 2.8% (15.24 mg/L, 0.43) ii) Phenytoin [2.5–40 mg/L] between run = 8.0% (5.18 mg/L, 0.42), 5.8% (15.03 mg/L, 0.83), 4.5% (30.40 mg/L, 1.36) and within run = 2.1% (5.00 mg/L, 0.11), 1.5% (13.76 mg/L, 0.20), 2.9% (28.19 mg/L, 0.80) iii) Valproic acid [10.0–150.0 mg/L] between run = 7.8% (33.26 mg/L, 2.59), 7.5% (75.90 mg/L, 5.68), 7.6% (125.70 mg/L, 9.55) and within run = 2.5% (33.03 mg/L, 0.82), 1.0% (67.99 mg/L, 0.70), 3.6% (112.83 mg/L, 4.09).
Quantitation was based on one 6-mm diameter DBS punch from the centre of the spot, containing approximately 15 µL of blood. AEDs extraction was performed using 500 µL of analytical grade (99%) acetonitrile (Prime Products Pte. Ltd., Singapore) and 1 molar sodium hydroxide (JT Baker, Phillipsburg, NJ, USA) at a ratio of 24∶1, v/v with 1 µg/mL 5-(p-methylphenyl)-5-phenylhydantoin (5MP) (Sigma Aldrich, St Louis, MO) as internal standard. The extraction procedure involved 1 min of vortexing and 5 min of sonication. Then, the mixture was centrifuged for 15 min at 6000
The analytical assay was developed and validated with a GC-MS system that comprised of GC 2010 Shimadzu GC coupled to a GCMS-QP2010 Plus quadrupole MS (Shimadzu Corporation, Nishinokyo-Kuwabara-cho, Nakagyo-ku, Kyoto, Japan). GCMSsolution (version 2.0), was utilized for data acquisition and peak area computation. DB5 ms (30 m×0.25 mm×0.25 µm) supplied by Agilent Technologies J&W, Inc. was used as the capillary column. Injector temperature was set at 250°C, while ion source at 220°C. Injection volume of 1 µL was subjected to split ratio of 1∶5 and column flow was set at 1.9 mL/min. Column temperature began at 90°C with a 0.2-min isothermal hold. Temperature was then ramped at 4 different rates: i) 10°C/min to 120°C, held for 0.5 min ii) 65°C/min to 285°C, held for 0.5 min iii) 10°C/min to 291°C, held for 0.2 min and iv) 60°C/min to 300°C for a final isothermal hold of 5 min. Selective ion monitoring (SIM) mode was used for detection of the target analytes at their respective retention times and are tabulated in
Analytes | Retention Time | Quantifying Ion | Qualifying Ions |
Valproic Acid | 3.453 min | 201 | 129, 145 |
Phenytoin | 7.068 min | 281 | 165, 176, 253 |
Carbamazepine | 7.166 min | 193 | 194, 293 |
5-methylphenylhydantoin | 7.245 min | 267 | 290, 395 |
Calibration and quality control standards were prepared in blood and spotted onto the 903 cards at 30 µL each. One 6-mm diameter disc was punched out from each DBS and used for analysis. The assay was validated over a range of 0.5–120 µg/mL for all three AEDs. Accuracy ranged from 100–110% and imprecision was <10%. The calculated limit of detection was 0.05 µg/mL for VPA and approximately 0.07 µg/mL for both PHT and CBZ. The recoveries of analytes were relatively high (75%–97%) and consistent (SD≤8.2%). Although the inconsistency increased to 11% at the lower limit of quantitation for CBZ, it was still within the FDA acceptable lower limit of quantification (<20%) (
DBS concentrations (Cdbs) and plasma concentrations (Cplasma) determined by the respective methods were directly compared. Theoretical Cplasma was calculated using a formula which accounts for the individual hematocrit values and red blood cell-to-plasma partition (RBC/plasma) ratio
Statistical analyses were done using SPSS 21 and MedCalc Version 12.6.1.0. Simple Deming regression was utilized to compare the 2 methods. The differences between methods were assessed using paired sample t-test. Bland-Altman plots were subsequently compiled using the theoretical Cplasma and observed Cplasma. Outliers were confirmed using standardized score and removal was considered if the score exceeded 2.5.
A total of 181 PWE were recruited but only 165 PWE who provided DBS were included in the final analyses. Fourteen PWE were excluded due to undetectable Cplasma (<2 µg/mL), and 2 PWE were excluded as outliers. Characteristics of PWE within each AED group are tabulated in
Characteristics | Valproic Acid (n = 92) | Phenytoin (n = 49) | Carbamazepine (n = 108) |
Number of Eligible DBS | 85 | 43 | 101 |
Number of Excluded DBS | 7 | 6 | 7 |
Number of Subjects | 84 | 41 | 100 |
Male | 48 (57.1%) | 22 (53.7%) | 48 (48%) |
Age, Median (Range) | 44.3 (19–78) | 50.6 (18–72) | 42.9 (20–78) |
Ethnic, No. Subjects (%) | |||
Chinese | 74 (88.1%) | 37 (90.2%) | 89 (89%) |
Malay | 5 (6.0%) | 3 (7.3%) | 5 (5%) |
Indian | 3 (3.6%) | nil | 6 (6%) |
Others | 2 (2.3%) | 1 (2.4%) | nil |
Concurrent medications, No. Subjects (%) | |||
None | 7 (8.3%) | 22 (53.7%) | 22 (22%) |
Valproic Acid | - | 10 (24.4%) | 46 (46%) |
Phenytoin | 10 (11.9%) | - | nil |
Carbamazapine | 46 (54.8%) | nil | - |
Other AEDs | 21 (25.0%) | 9 (21.9%) | 32 (32%) |
Blood Chemistry, Median (range) | |||
Hematocrit (%) | 41.6 (30.7–51) | 42.7 (33.3–49.2) | 41.3 (29.8–49.7) |
Hemoglobin (g/dL) | 13.9 (9.8–16.8) | 14.0 (10.5–16.4) | 13.6 (9.2–16.8) |
Liver Function Test | Median (range) | ||
Albumin (g/L) | 40 (31–46) | 41 (31–47) | 41 (31–46) |
ALT (U/L) | 20 (8–109) | 24 (12–79) | 19 (9–43) |
AST (U/L) | 22 (12–59) | 22 (16–78) | 21 (12–59) |
GGT (U/L) | 53 (9–333) | 84 (27–417) | 50 (21–213) |
Drug Monitoring (µg/mL), Mean (standard deviation) | |||
Mean plasma levels | 57.1 (22.35)* | 9.7 (4.67)* | 8.4 (2.32) |
Mean DBS levels | 29.2 (14.67)* | 6.9 (3.92)* | 8.3 (2.56) |
Mean predicted plasma levels | 57.1 (20.31) | 9.7 (4.60) | 8.4 (2.27) |
Average dose (mg), Mean (standard deviation) | |||
870.5 (413.22) | 934.0 (317.89) | 284.1 (71.33) | |
Drug Responsive | 28 (33.3%) | 21 (51.2%) | 31 (31%) |
Drug Resistant | 31 (36.9%) | 9 (22.0%) | 38 (38%) |
Undefined | 25 (29.8%) | 11 (26.8%) | 31 (31%) |
Total recruited PWE were 183. Only 169 were included in the analysis. The remaining 14 subjects were excluded due to missing plasma levels from hospital laboratory system. (Note: Some recruited PWE contributed to the levels of two AEDs).
Plasma concentrations of (
When regressed against the theoretical Cplasma, definite improvements in fit between data points from the turbimetric assay and DBS assay were observed. The lines of regression for respective AEDs rotated closer to the line of unity and data points clustered in greater proximity along the identity line, albeit marginal decrease in correlation coefficients for PHT and VPA (
Plasma concentrations of (
Bland Altman plots for plasma concentrations of (
In line with a presumed K of 1.06 from literatures, the RBC dilutional effect on Cdbs of CBZ was found to be negligible
Contrasting to CBZ, the Cdbs of VPA was found to be constantly lower than its Cplasma. Similar finding were previously observed by Vermeij and Edelbroek in their study. They found a conversion factor of 1.46 from Cdbs to Cplasma, which implied that the Cplasma of VPA was 46% higher than its Cdbs
After incorporating the hematocrit and K values, definitive improvement was achieved for the computation of the theoretical Cplasma of VPA. Yet, the theoretical Cplasma derived from Cdbs remained consistently lower than Cplasma. The most probable explanations include the domination of RBC dilutional effect and VPA binding to 903 cards. In clinical practice, however, the potential consequence of the difference in calculated Cplasma from Cdbs may not be obvious. An example of a worst case prediction would be, for an actual Cplasma of 60 µg/mL, the predicted Cplasma could either be 48 or 72 µg/mL. Regardless of the variation in the predicted Cplasma, physicians are likely to increase the dose if the PWE has uncontrolled seizure, hence arriving at the similar clinical decisions. Therefore, the clinical decisions premised upon Cdbs will be no different from the clinical decisions made using Cplasma.
On the other hand, PHT is readily partitions into and dissociates from the RBC, with a K reported to be approximately 0.29
In this study, we demonstrated that compound-specific RBC-to-plasma binding and individual hematocrit level could explain the difference in concentrations detected from DBS and plasma. It seems that as RBC-to-plasma partitioning approaches 1, e.g. 1.06 for CBZ, the closer the Cdbs is to its Cplasma and hematocrit level will have no dilution effect. Conversely, as RBC-to-plasma partitioning approaches 0, the dilution effect of hematocrit level becomes prominent and higher hematocrit levels lower Cdbs. DBS does seem to equate the whole blood characteristics which were demonstrated in previous studies
At clinically relevant blood and liver biochemistry variations, correcting the Cdbs to hematocrit and K improved the theoretical prediction of Cplasma for PHT and VPA. Nevertheless, this study catered for the investigation of PWE with AED binding to the red blood cells and albumin in the normal ranges. It is reasonable to assume that the effects of AED binding to blood cells and albumin should not fluctuate significantly. For the former effect, the Ks were fixed at 2 values for PHT and VPA. As evidenced by the improved graphical fit, correspondence with the actual Cplasma was improved with the inclusion of K. Similary, a constant K value has also been used to enhance the clinical applicability and harmonize the analytical process of PHT and VPA in whole blood samples
The PWE recruited in this study represented the typical population of PWE who required the routine therapeutic drug monitoring. However, there were no inclusion of children, critically ill patients nor subjects who have just started the AED/s treatment. Hence, the applicability of this DBS quantitation method for dose titration for these cohorts of subjects remains unknown. These subjects could have a more fluctuating Hct and/or drug levels. Their theoretical Cplasma would be more challenging to be determined from the corresponding Cdbs levels. The applicability of Cdbs as a surrogate to Cplasma and even seizure control in these subjects need to be established in the future studies.
It is noteworthy that on this study, the DBS was obtained from venous source. Theoretically, there could be some differences between capillary and venous concentrations but the differences for a majority of xenobiotics were proven not to be obvious, especially after the distribution phase
In view of the good agreement between the theoretical Cplasma estimated from the Cdbs levels and the observed Cplasma for PHT and VPA, and also between the Cdbs and the observed Cplasma for CBZ, DBS is deemed suitable as an alternative matrix to the conventional plasma samples for TDM of AEDs in the adult population of PWE. Further studies that investigate the pharmacokinetic parameters such as clearance and apparent volume of distribution using the Cdbs levels and then correlate these concentrations to the treatment outcomes are warranted.
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Previous presentation: Poster presentation of partial results at 10th European Congress on Epileptology, London, 30 September – 4 October 2012.