[Determination of morphine, codeine and 6-monoacetylmorphine in saliva of substance-abuse patients using HPLC/MS methods].

BACKGROUND/AIM: Saliva represents an alternative specimen for substances abuse determination in toxicology. Hence, the aim of this study was to optimize a method for saliva specimen preparation for heroin metabolites, morphine and 6-monoacetylmorphine (6-mam), and codeine determination by liquid chromatography-mass spectrometry (LC/MS), and to apply this method on saliva samples taken from the patients. METHODS: Saliva specimen was prepared using liqiud/liquid extraction of morphine, codeine and 6-mam by mixture of chloroform and isopropanol (9 : 1; v/v). Extracts were analysed by HPLC/MS technique: separation column Waters Spherisorb 5 microm, ODS2, 4.6 x 100 mm; mobile phase: ammonium acetate : acetonitile (80 : 20; v/v), mobile phase flow rate 0.3 mL/min; mass detection range: 100-400 m/z. Regression and correlation analyses were performed with the probalility level of 0.05. Concentrations of morphine, codeine and 6-mam were determined in saliva samples of the patients with "opiates" in urine identified by the test strips. RESULTS: Calibration for each analysed substance was done in the concentration range from 0.1 to 1 mg/L and the coefficient of correlation was R2 > 0.99. We obtained following calibration curves: y = 385531x + 14584; y = 398036x + 31542; and y = 524162x - 27105, for morphine, codeine and 6-mam, respectively. Recovery for morphine and codeine determination was 99%, while for 6-mam it was 94%. Limits of detection and quantification of a proposed method were 0.01 mg/L and 0.05 mg/L, respectively. Concentration of morphine in the saliva of the heroin users ranged between 0.54 and 5.82 mg/L, concentration of codeine between 0.05 and 5.33, and 6-mam between 0.01 and 0.68 mg/L. A statistically significant correlation between codeine and 6-mam concentrations was obtained. CONCLUSION: A proposed HPLC/MS method for morphine, codeine and 6-mam determination in saliva is accurate, simple, cheap and suitable for routine analysis and monitoring of heroin abuse.

[Simultaneous determination of amoxicillin and clavulanic acid in the human plasma by high performance liquid chromatography-mass spectrometry (UPLC/MS)].

BACKGROUND/AIM: Quantitative analysis of amoxicillin and clavulanic acid in biological matrices requires sensitive and specific methods which allow determination of therapeutic concentration in 1 g/mL range. Analytical methods for determination of their concentrations in body fluids described in literature include high performance liquid chromatography coupled to UV detector (HPLC-UV) and liquid chromatography-mass spectrometry (LC-MS). The aim of this study was to develop sensitive and specific ultra performance liquid chromatography/mass spectrometry (UPLC/MS) method which could be used for the spectral identification and quantification of the low concentrations of amoxicillin and clavulanic acid in the human plasma. METHOD: A sensitive and specific UPLC/MS method for amoxicillin and clavulanic acid determination was developed in this study. The samples were taken from the adult healthy volunteers receiving per os one tablet of amoxicillin (875 mg) in combination with clavulanic acid (125 mg). RESULTS: Plasma samples were pretreated by direct deproteinization with perchloric acid. Quantification limit of 0.01 microg/ml for both amoxicillin and clavulanic acid was achieved. The method was reproducible day by day (RSD < 7%). Analytical recoveries for amoxicillin ranged from 98.82% to 100.9% (for concentrations of 1, 5 and 20 microg/mL), and recoveries for clavulanic acid were 99.89% to 100.1% (for concentrations of 1, 2 and 5 microg/mL). This assay was successfully applied to a pilot pharmacokinetic study in healthy volunteers after a single-oral administration of amoxicillin/clavulanic combination. The determined plasma concentrations of both amoxicillin and clavulanic acid were in the range of the expected values upon the literature data for HPLC-UV and LC-MS methods. CONCLUSION: The described method provided a few advantages comparing with LC/MS-MS method. The method is faster using running time of 5 minute, has lower limit of quantification (LOQ) and it could be used in pharmacokinetic studies of both amoxicillin and clavulanic acid.

A randomized, open-label pharmacokinetic comparison of two oral formulations of fluconazole 150 mg in healthy adult volunteers.

BACKGROUND: Because of its systemic action, fluconazole is prescribed for a variety of fungal infections. However, therapeutic failure might result when a patient is switched between an innovator drug and a nonbioequivalent generic formulation. Pharmacokinetic (PK) studies investigating the bioequivalence of generic and innovator drugs can minimize such risks. OBJECTIVE: The aim of this study was to compare the PK profiles and relative bioavailabilities of 2 oral formulations of fluconazole: Diflucan (reference; Pfizer Corporation Austria GmbH, Wien, Austria) and Funzol (test; Bosnalijek d.d., Pharmaceutical and Chemical Industry, Sarajevo, Bosnia and Herzegovina), both prepared as capsules containing 150 mg of active drug. METHODS: A single oral dose of fluconazole was given under fasting conditions to healthy, white volunteers aged 18 to 55 years in this open-label, randomized, crossover study. A 3-week washout period was applied between each of the 2 doses. Serum samples were obtained before dosing and at various time points after dosing up to 144 hours and were analyzed for fluconazole concentration using a high-performance liquid chromatography-UV method. PK parameters representing the extent (AUC(0-infinity)) and rate (CmaX and T(max)) of absorption of fluconazole were obtained. An analysis of variance, a power analysis, 90% CI, and two 1-sided tests were used for statistical analysis of relative differences between the 2 drugs. Bioequivalence was concluded if the 90% CIs for the geometric mean ratios of AUC(0-infinity) and C(max) were between 0.80 and 1.25. A study investigator monitored the volunteers for adverse effects at 5 defined time points during the clinical part of the investigation. RESULTS: Thirteen men and 11 women (mean age, 33.3 years; mean weight, 73.6 kg) completed the study. The respective point estimates of the ratios of geometric means of log-transformed C(max) and AUC0(0-infinity) of fluconazole (test vs reference) were 0.985 and 1.047, with 90% CIs of 0.894 to 1.085 and 0.927 to 1.182, respectively. Differences in T(max) also did not reach statistical significance. No adverse effects were reported by the subjects or revealed by clinical or laboratory tests. CONCLUSIONS: The study failed to demonstrate any statistically significant differences in C(max) and AUCO(0-infinity) values between the test and reference formulations of oral fluconazole 150 mg in this small, select population of healthy volunteers. On that basis, and according to both the rate and extent of absorption, the test and reference formulations were considered bioequivalent.