人血浆中磺胺甲噁唑和代谢产物的HPLC法测定及其药代动力学研究

作者                        作者单位

李向阳                青海大学医学院药学系

朱俊博                青海大学医学院药学系

格日力                青海大学医学院高原医学研究中心

1 Introduction

Sulfamethoxazole is an antibacterial sulfonamide and is now used primarily to treat urinary tract infection, meningitis, respiratory tract infection and dermatitis glandularis erythematosa in combination with trimethoprim, a combination product known as Bactrim[1，2], but it can cause a serial of adverse effect such as allergic response, nausea, emesis, hepatotoxicity, hematology toxicity, mucocutaneoocular syndrome and so on after long term use[3]. Sulfamethoxazole is 6070% proteinbound with an extent of absorption of 90% and is metabolized primarily by Nacetyltransferase 2 (NAT2) as N4acetylsulfamethoxazole in liver, and it’s toxicity related to it’s metabolites, metabolic pathway and acetylator phenotype [4，5]. The pharmacokinetics of sulfamethoxazole in healthy subjects was reported in previous studies [6，7], but little information is available on the pharmacokinetic properties of N4acetylsulfamethoxazole in Chinese subjects. In this study, we established a highly sensitive and rapid assay method by using RPHPLC in order to study pharmacokinetics of sulfamethoxazole and metabolite N4acetylsulfamethoxazole in human. The assay is validated over the range of 1 to 160 μg/ml for sulfamethoxazole and 0.5 to 20 μg/mL for it’s metabolite N4acetylsulfamethoxazole, and the method has been used successfully to study sulfamethoxazole and metabolite N4acetylsulfamethoxazole pharmacokinetics in humans.

2 Experimental

2.1 Materials and reagents

Complex sulfamethoxazole (Co SMZ) tablets were provided by Guangdong Baiyunshan pharmaceuticals of China. Sulfamethoxazole and N4acetylsulfamethoxazole were purchased from National Institute for the Control of Pharmaceutical and Biological Products of China and Toronto Research Chemicals Inc, respectively. All reagents and solvent used for the chromatography and sample procedures were of analytical or higher quality and were purchased from Baiyin Chemical Regent corporation, Gansu, China, Yuwang Chemical Regent corporation, Shandong, China. Purified water was purchased from Wahaha Corporation, Zhejiang, China.

2.2 Instrument and Conditions

HPLC analyses were performed using a SHIMADZU LC10AT and SPD10A system (SHIMADZU corpohumanion, Japan) with a Lichrospher C18 column (4.6mm×250mm, 5 μm, Jiangsu Hanbang Science and technology Co. China). The mobile phase was acetonitrilewateracetic acidtriethylamine (38:62:0.4:0.3, v/v/v/v). The column temperature was maintained at 25℃. A constant flowrate of 1.0 mL/min was employed throughout the analyses, and the detection was performed at UV 240 nm.

2.3 Preparation of standard solutions, Calibration standards, limit of quantitation (LOQ) and quality control samples

Stock solutions of sulfamethoxazole and N4acetylsulfamethoxazole was prepared at 1 mg/ml in methanol and stored at 4℃, respectively. Standard solutions containing 1 μg/ml, 10 μg/ml and 100 μg/ml were prepared by diluting the stock solution with methanol. These solutions were stable for 6 months at least.

Calibration standards of sulfamethoxazole (1, 5, 10, 20, 40, 80, 120, 160 μg/ml) and N4acetylsulfamethoxazole (0.5, 1, 2.5, 5, 10, 20 μg/ml) and a LOQ sample at 1 μg /ml for sulfamethoxazole and 0.5 μg /ml for N4acetylsulfamethoxazole were prepared by spiking appropriate amount of the standard solutions in control plasma obtained from healthy persons respectively. Quality control (QC) samples were prepared in blank control plasma at concentrations of 1, 40 and 160 μg/ml for sulfamethoxazole and 0.5, 5 and 20 μg /ml for N4acetylsulfamethoxazole.

2.4 Plasma sample preparation

To a 0.5ml aliquot of plasma in a 2ml centrifuge tube and 0.15 ml 30% perchloric acid was added. The centrifuge tube was vortexed for 1 min, and then centrifuged for 10 min in 16000 r/min. The 10 μl aliquot of supernatant was injected into the HPLC system.

2.5 Assay validation

2.5.1 Linearity and LOQ

Calibration standards of eight concentrations of sulfamethoxazole and N4acetylsulfamethoxazole and LOQ plasma sample of sulfamethoxazole and N4acetylsulfamethoxazole were assayed, respectively. A calibration curve was constructed by plotting the area of sulfamethoxazole and N4acetylsulfamethoxazole against concentrations in plasma. LOQ for sulfamethoxazole and N4acetylsulfamethoxazole was established based on an S/N ratio of 10.

2.5.2 Precision and accuracy

The precision of the assay was determined from the low, medium and high QC plasma samples by replicate analyses of the three different concentrations (1, 40 and 160 μg/ml for sulfamethoxazole, 0.5, 5 and 20 μg /ml for N4acetylsulfamethoxazole). Intraday precision was determined by repeated analysis of each QC sample on one day (n=5), and interday precision and accuracy was determined by repeated analysis on five consecutive days (n=1 series per day). The concentration of each sample was determined using calibration standards prepared on the same day. Accuracy is defined as the relative deviation in the computed value (E) of a standard from that of its true value (T) expressed as a percentage (RE %). It was calculated using following formula: RE %=(ET)/T·100. Assay precision was defined as the relative standard deviation (SD) from the mean (M), calculated using the equation RSD %=SD/M·100%.

2.5.3 Absolute recovery

The absolute recovery of sulfamethoxazole and N4acetylsulfamethoxazole was determined at low, medium and high concentrations by the external standard method. A known amount of sulfamethoxazole and N4acetylsulfamethoxazole was added to plasma prior to plasma sample preparation as described in section 2.4. Concentration of sulfamethoxazole and N4acetylsulfamethoxazole was calculated using the calibration curves prepared on the same day, and was compared to the nominal concentration to estimate recovery.

2.6 Pharmacokinetics and study

Each volunteer received a single oral dose of 3 tablets of complex sulfamethoxazole containing 1,200 mg of sulfamethoxazole with 150 ml of water at 8:00 AM after fasting overnight (12 hours) with water allowed ad libitum. Serial blood samples (3 ml each) were collected from an indwelling venous catheter (ID:1.3 mm, length:19 mm) into heparinised tubes before (baseline) and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36 and 48 h after starting drug administration. Food or beverages containing alcohol or caffeine were not allowed from 48 h before drug administration until the last blood sample was drawn. Plasma were separated by centrifugation (4,000 g for 10 min at 4 ℃) and immediately stored at 20 ℃ until the quantitative analysis of drugs in plasma. Noncompartmental analysis using DAS 2.0 software (Institute of Clinical Pharmacology, Wannan medical college, China) was performed to calculate the absorption rate constant (Ka), elimination rate constant (Ke), clearance (CI), volume of distribution (Vd), mean residence time (MRT), plasma halflife (t1/2), and area under curve (AUC) for sulfamethoxazole and N4acetylsulfamethoxazole, respectively. The peak plasma concentrations (Cmax) and the time of maximum concentration (tmax) were obtained directly from the raw data.

3 Result and discussion

3.1 Conditions of chromatography

The selection of mobile phase components was a critical factor in achieving good chromatographic peak shape and resolution. In this solvent system, acetic acid was selected as a regulator for its good volatility. Moreover, acetic acid could inhibit the tailing problem of chromatographic peaks of sulfamethoxazole and N4acetylsulfamethoxazole. Good separation of target compounds and short run time were obtained by using an elution system of acetonitrilewateracetic acidtriethylamine (38:62:0.4:0.3, v/v/v/v). Representative chromatograms are shown in Fig. 2 in which the retention times of sulfamethoxazole and N4acetylsulfamethoxazole were 5.7 and 4.3 min, respectively.

Fig 2. Chromatograms of control solution (A), blank plasma solution(B), blank plasma solution added Sulfamethoxazole and N4acetylsulfamethoxazole (C) and plasma solution after administration (D) (peak 1: Sulfamethoxazole; peak 2: N4acetylsulfamethoxazole)

3.2 Method validation

3.2.1 Calibration curve and sensitivity

The calibration curves which relate the concentrations of sulfamethoxazole and N4acetylsulfamethoxazole to their area were linear over the range of 1 to 160 μg/ml for sulfamethoxazole and 0.5 to 20 μg/ml for N4acetylsulfamethoxazole. A typical calibration curve had a slope of 7768, an intercept of 1584 and R = 0.9998 for sulfamethoxazole, and a slope of 1009, an intercept of 961 and R = 0.9998 for N4acetylsulfamethoxazole. A calibration curve was prepared contemporaneously with each batch of samples. LOQ for sulfamethoxazole and N4acetylsulfamethoxazole in plasma, defined at a N/S = 10:1, were 1 and 0.5 μg/ml, respectively.

3.2.2 Precision and accuracy

The batch to batch coefficient of variation of spiked quality control samples was 8.4%, 2.9% and 2.1% at sulfamethoxazole concentrations of 1, 40 and 160 μg/mL and 9.6%, 4.4% and 4.2% at N4acetylsulfamethoxazole concentrations of 0.5, 5 and 20 μg/mL, respectively. The accuracy was calculated as the differences between given and measured mean concentration, expressed as a percentage, and was 6.0%, 4.1% and 5.7% (sulfamethoxazole) and 10.3%, 0.9% and 1.2% (N4acetylsulfamethoxazole) at the abovementioned target concentrations.

3.3 pharmacokinetics parameters

The method described above was successfully applied to the pharmacokinetic study in which plasma concentrations of sulfamethoxazole and N4acetylsulfamethoxazole in 20 healthy persons were determined up to 48 h after administhumanion of complex sulfamethoxazole containing 1,200 mg of sulfamethoxazole. The mean plasma concentrationtime curve is shown in Fig.3. The pharmacokinetic parameter values are calculated and shown in Table 3.

4 Discussion

It is considered that the method was sensitive, accurate and simple for the determination of sulfamethoxazole and it’s metabolite N4acetylsulfamethoxazole in human plasma，it has been used successfully to study pharmacokinetics of sulfamethoxazole and it’s metabolite in adult healthy persons.

In this study, the pharmacokinetic parameters t1/2, ke, Tmax and CL found in Chinese healthy male volunteers are agreed with those reported in the recent pharmacokinetic studies of sulfamethoxazole[7] in which a MRT of approximately 14–18 h has been reported, but the value for MRT in our study was approximately of 12 h. The pharmacokinetic parameters t1/2, ke, and CL of N4acetylsulfamethoxazole is also in accord with this earlier study, however, Tmax is 30% lower in our study in comparison with their study.

Sulfamethoxazole is converted mainly to N4acetylsulfamethoxazole by Nacetyltransferases in humans, and the elimination of N4acetylsulfamethoxazole is a formation ratelimited metabolism. Sulfonamides are associated with a variety of adverse reactions, some of which have been linked with the classical acetylator phenotypes. Although the slow acetylator phenotype has been identified as a risk factor for hypersensitivity reactions to sulfamethoxazole, the disposition of this compound appears not to be affected by the acetylation polymorphism in vivo in humans [8]. Slow and fast acetylators for sulfamethoxazole could not be distinguished in this study.

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