Determination of carbamazepine in serum and saliva samples by high performance liquid chromatography with ultraviolet detection

Background/Aim. Carbamazepine is antiepileptic drug widely used for the treatment of epilepsy. Due to low therapeutic index of carbamazepine there is a need for routine measuring its concentrations in biological fluids. The aim of the study was to describe a method for concomitant determination of carbamazepine in the serum and saliva. Methods. Separation of the drug from matrix is achieved by reversedphase chromatography on a C18 column, with a mobile phase of methanol-water-acetic acid (65:34:1) at a flow-rate of 1.0 ml/min. Detection was effected by ultra-violet absorption at 285 nm. The total run time was 5 min. Samples were prepared by alkaline extraction (pH 10) using chlorophorm. Results. Calibration curves were in the range 0.1–5 μg/mL for serum and saliva samples. Mean recoveries of spiked serum and saliva were 97.59 and 92.30%, respectively. Limits of detection (LOD) of carbamazepine in serum and saliva were 0.166 and 0.178 μg/mL, respectively. Limits of quantification (LOQ) in the serum and saliva were 0.237 and 0.226 μg/mL, respectively. The method precision was carried out with coefficient of variation of 2.10% and 4.03% for the serum and saliva, respectively. The obtained data showed that there was a strong correlation between saliva and serum concentrations (r = 0.9481, p < 0.001). Conclusion. The method described here is rapid, precise, accurate and simple, and can be used for quantitative determination of carbamazepine in human serum and saliva after therapy applying. Saliva samples could be used as an alternative matrix for therapeutic drug monitoring of this antiepileptic drug.


Introduction
Carbamazepine is an iminostilbene derivative used for more than three decades as the drug of first choice for the treatment of trigeminal neuralgia and also for both generalized and partial seizures, due to rapid control of excessive cerebral electrical discharges and the low incidence of acute and chronic toxicity 1 .
Carbamazepine is almost entirely metabolized by the CYP450 enzyme system in the liver.It induces cytochrome P450 isoenzymes as well as UDP-glucuronyltransferase and may also inhibit CYP2C19.Carbamazepine undergoes autoinduction (via CYP3A4).Certain drugs (cimetidine, diltiazem, verapamil, erythromycin) can increase carbamazepine serum levels.On the other hand, drugs that accelerate hepatic metabolism (phenytoin, phenobarbital, primidone, oxcarbazepine) will decrease serum concentrations of carbamazepine.Taking carbamazepine simultaneously with lamotrigine may increase the likelihood of neurotoxic side effects 2 .Because of its low therapeutic index and increasing number of carbamazepine intoxications there is a need for routine measuring of carbamazepine concentration in serum samples.
The most applying methods for determination of carbamazepine in biological materials use high performance liquid chromatography with ultraviolet or photodiode detection (HPLC-UV, HPLC-PDA) and immunoassay (FPIA) 1, 3- 11 .Gas chromatography with mass spectrometry and liquid chromatography with mass spectrometry methods have been described in the literature 12,13 .
The aim of this paper was to describe a simple, accurate and precise HPLC-UV method for determination of carbamazepine in serum and saliva samples.

Methods
Analytical standard of carbamazepine (100% s.s.) was obtained from Sigma.
Methanol and glacial acetic acid were of HPLC and p.a. purity, obtained from MERCK.Ammonium hydroxide 25% was p.a. purity and obtained from J. T. Baker.Water was purified by Millipore Milli-Q system.
The method used was a high performance liquid chromatograph LKB 2150 binary pump with Waters 2487 dual λ Absorbance Detector and Clarity Lite Software.
Stock standard solution of carbamazepine was prepared by dissolving 10 mg in 10 mL methanol and stored at -4ºC.Other concentrations of carbamazepine were made by diluting stock standard solutions with mobile phase to achieve calibration concentrations expected to meet the therapeutic levels in serum of patients.
To 0.2 mL serum and 1 mL saliva samples, 50 μL of 25% ammonium hydroxide and 5 mL of chloroform was added.The samples were mixed in a mechanical shaker for 20 minutes and centrifuged at 3 000 rpm for 10 min.After centrifugation, organic layer was separated and evaporated in a stream of air.Dry extracts were reconstituted in mobile phase and analyzed by HPLC-UV method on 285 nm.
Calibration and quality control samples were prepared by adding carbamazepine solution in blank ("drug-free") human serum and saliva.The amounts corresponded to the serum and saliva concentrations of carbamazepine ranged from 0.1 to 5 μg/mL.The calibration curves for the saliva and serum spiked by carbamazepine were obtained by plotting carbamazepine peak areas for the concentrations range 0.1, 0.5, 0.75, 1, 1.5, 2.5 and 5 μg/mL.
The concentrations of quality control samples were 0.1, 1 and 5 mg/L.

Results
Carbamazepine concentrations were determined using weighed linear regression function.Table 1 shows a calibration curve for carbamazepine in the serum and saliva.
The correlation coefficients for the saliva and serum spiked by carbamazepine were 0.9969 and 0.9996, respectively.Figure 1 shows a calibration curve for spiked serum and saliva.
Unknown concentrations of carbamazepine in serum and saliva samples of the patients were calculated using a corresponding factor from the calibration curve.A factor is calculated from the mean value peak-area of carbamazepine for each mentioned concentration.Recovery was determined for each concentration, as the mean of three samples by comparing the peak areas of the extracted and non-extracted samples.
Figures 2a to 2d show chromatograms of drug-free serum and saliva and serum and saliva spiked by carbamazepine solution concentration of 1 mg/L.
A precision of the method was assessed by calculating coefficient of variation (CV) for a measured parameter of the method (peak area of carbamazepine) and determined on the same day (n = 7).The inter-and intra-day coefficients of variation (CV) for spiked serum and saliva are shown in Table 2.
The spiked saliva and serum extracts were stable when kept in a refrigerator for 48 hours; mean 97.36% (95.35 -103.02%).Carbamazepine in serum and saliva was stable following 3-fold freezing and thawing (F&T) procedure.The results are shown in Table 3.
Calculation of carbamazepine concentration was done on the basis of the calibration curve obtained after analysis of saliva and serum spiked by carbamazepine standard solution ranged from 0.1 to 5 mg/L.Linear regression for spiked saliva and serum were Y = 1843.9*X+ 151.84 and Y = 1874.1*X-8.46, respectively.
Serum and saliva constituents did not interfere in the carbamazepine assay.
The method was applied to determine a concentration of carbamazepine in the patient`s serum or saliva samples.We calculated serum/plasma (S/P) ratio on the basis of the determined salivary and serum carbamazepine concentrations.
Table 5 shows salivary and serum carbamazepine concentrations and S/P ratio of 23 patients on the antiepileptic therapy.

Discussion
There are different methods for sample preparation for carbamazepine determination.Precipitation of proteins from serum samples preparation has been cited in the literature 14 .Also, liquid-liqiud extraction of carbamazepine by methyl tert-butyl ether, ethyl acetate and ether have been described 8,15,16 .
Carbamazepine could be determined by the HPLC-UV method after solid phase extraction on C18 or Oasis HLB cartridges [17][18][19] .
We performed liquid-liquid method for the extraction of carbamazepine from serum and saliva samples by chloroform at pH 10.This extraction gave very good recoveries for serum and saliva spiked by carbamazepine: 97.59% (from 87.85% to 102.49%) and 92.30% (from 89.25 to 96.84%), respectively.Liquid-liquid extraction is necesery for preparation of saliva samples because of its high viscosity.So, in that case solid phase extraction is not possible.The same ex- We had performed separation of carbamazepine from matrix compound on C18 column as many other authors [14][15][16][17][18][19][20][21] .
The HPLC-UV method mostly used mobile phase with phosphate buffer [21][22][23] .Six antiepileptic drugs were eluted from C18 column with mobile phase consisting of a 0.01 M phosphate buffer-methanol-acetonitrile (65:18:17 v/v) adjusted to pH 7.5 with phosphoric acid.The effluent was monitored at 220 nm 16 .
Shimoyama et al. 18 described chromatographic separation on C8 column using a mobile phase of potassium dihydrogenphosphate (pH 2.5) and acetonitrile (67:33 v/v) with UV detection on 254 nm.
We used mobile phase contained methanol-waterglacial acetic acid (65:34:1).The content of our mobile phase was similar to the mobile phase that contained methanolwater-glacial acetic acid (67 : 33 : 1) applied in the HPLC-UV method for determination of carbamazepine described by Kishore et al. 15 .Applying this mobile phase we obtained good sensitivity.The compounds of matrix did not interfere with analyte (Figures 2a and 2b).
Different wavelengths for detection of carbamazepine such as 210 nm 8,20,21 , 220 nm 16 , 230nm 15 or 254 nm 18 have been reported in the literature.
We used the wavelength of 285 nm allowing carbamazepine maximum of absorbance and the best sensitivity of the method.Moreover, the compounds of matrix could be interfering with carbamazepine at the lower wavelengths such us 220 or 230 nm.
Limit of quantitation for carbamazepine described by Levert at al. was 0.66 mg/L 14 .Quantification limit for carbamazepine after single-step protein precipitation procedure and detection on 215 nm was 0.5 mg/L 21 .
Limits of detection of the described HPLC-UV method in the serum and saliva were 0.055 and 0.054 mg/L, respectively.Limits of quantification of carbamazepine in the serum and saliva were 0.168 and 0.173 mg/L, respectively.In comparison with HPLC methods cited in the literature our method showed higher sensitivity with a lower limit of quantification 21 .
Leuert et al. developed a HPLC method for simultaneous determination of oxcarbazepine, carbamazepine and metabolites, phenobarbital and phenytoin in the serum.Mean recovery of this method after precipitation of serum samples was 96.52% 5 .
The recoveries of HPLC method for determination of carbamazepine and carbamazepine-10,11-epoxide in the plasma after solid-phase extraction on C18 cartridges were 88.9-104.0% 18.Our data were similar with the data in the literature.Analytical recoveries for the serum and saliva spiked by carbamazepine in the method that we developed were 97.59% (from 87.85% to 102.49%) and 92.30 % (from 89.25 to 96.84%), respectively.
The HPLC-UV method for simultaneous determination of carbamazepine, lamotrigine, primidone, phenobarbital and phenytoin used mobile phase consisting of water/ACN/methanol/triethylamine in the ratio 72 : 23 : 5 : 0.1 adjusted to pH 7.0.UV detection was carried out on 220 nm.The method was linear in the range of 1.25 -25 mg/L for carbamazepine.Within-day CV% and between-day CV% were within 10% 6 .
A HPLC assay with UV detection developed for the simultaneous determination of the anti-epileptic drugs lamotrigine, carbamazepine and zonisamide in human plasma and serum was linear at the range of 2 -20 mg/L for carbamazepine 7 .
Our method was linear in the range of 0.1 to 5 mg/L of carbamazepine in both serum and saliva.This range was in compliance with therapeutic range of the drug in serum and saliva samples.
The method was found to be reproducible with the coefficient of variation of 2.10% and 4.03 % for the serum and saliva, respectively.
The method was applied to analyze serum and saliva samples of patients on carbamazepine therapy and to determine if a correlation of drug concentrations between the saliva and serum exists.Our results show that a mean S/P ration is 0.39 (from 0.24 to 0.52) and that there is a strong correlation (r = 0.9481, p < 0.001) between the saliva and serum.
The mean ratio of salivary to serum free carbamazepine concentrations of 1.02 ± 0.11 has been reported 22 .However, other authors, similarly to us, have reported the saliva/plasma ratio for carbamazepine of 0.13 to 0.33 and intra-individual variation about 5% 22 .We consider that our results are acceptable because we measured no free, but total carbamazepine concentration in the serum (free carbamazepine and carbamazepine bounded to serum proteins).
The findings of this study demonstrate that monitoring of salivary carbamazepine concentrations, the most used anti-epileptic drug, proved to be a realistic alternative in routine clinical analysis after therapy applying especially in pediatric patients, because saliva collection is painless, noninvasive and simpler than blood taking.

Conclusion
The HPLC-UV method described here is rapid, sensitive and simple for determination of carbamazepine in human serum and saliva samples.Saliva samples could be used as an alternative matrix for therapeutic drug monitoring of this antiepileptic drug.