In situ derivatization hollow fibre liquid-phase microextraction for the determination of biogenic amines in food samples

Hollow fibre liquid-phase microextraction with in situ derivatization using dansyl chloride has been successfully developed for the high-performance liquid chromatography-ultraviolet (HPLC-UV) determination of the biogenic amines (tryptamine, putrescine, cadaverine, histamine, tyramine, spermidine) in food samples. Parameters affecting the performance of the in situ derivatization process such as type of extraction solvent, temperature, extraction time, stirring speed and salt addition were studied and optimized. Under the optimized conditions (extraction solvent, dihexyl ether; acceptor phase, 0.1 M HCl; extraction time, 30 min; extraction temperature, 26 °C; without addition of salt), enrichment factors varying from 47 to 456 were achieved. Good linearity of the analytes was obtained over a concentration range of 0.1–5 μg mL⁻¹ (with correlation coefficients of 0.9901–0.9974). The limits of detection and quantification based on a signal-to-noise ratio of 3–10, ranged from 0.0075 to 0.030 μg mL⁻¹ and 0.03 to 0.10 μg mL⁻¹, respectively. The relative standard deviations based on the peak areas for six replicate analysis of water spiked with 0.5 μg mL⁻¹ of each biogenic amine were lower than 7.5%. The method was successfully applied to shrimp sauce and tomato ketchup samples, offering an interesting alternative to liquid–liquid extraction and solid phase extraction for the analysis of biogenic amines in food samples.     

Sensitive determination of fluoride in biological samples by gas chromatography–mass spectrometry after derivatization with 2-(bromomethyl)naphthalene

A gas chromatography–mass spectrometric method was developed in this study in order to determine fluoride in plasma and urine after derivatization with 2-(bromomethyl)naphthalene. 2-Fluoronaphthalene was chosen as the internal standard. The derivatization of fluoride was performed in the biological sample and the best reaction conditions (10.0 mg mL⁻¹ of 2-(bromomethyl)naphthalene, 1.0 mg mL⁻¹ of 15-crown-5-ether as a phase transfer catalyst, pH of 7.0, reaction temperature of 70 °C, and heating time of 70 min) were established. The organic derivative was extracted with dichloromethane and then measured by a gas chromatography–mass spectrometry. Under the established condition, the detection limits were 11 μg L⁻¹ and 7 μg L⁻¹ by using 0.2 mL of plasma or urine, respectively. The accuracy was in a range of 100.8–107.6%, and the precision of the assay was less than 4.3% in plasma or urine. Fluoride was detected in a concentration range of 0.12–0.53 mg L⁻¹ in six urine samples after intake of natural mineral water containing 0.7 mg L⁻¹ of fluoride.     

Simultaneous determination of salbutamol sulphate, bromhexine hydrochloride and etofylline in pharmaceutical formulations with the use of four rapid derivative spectrophotometric methods

Four simple, rapid, accurate, precise, reliable and economical spectrophotometric methods have been proposed for simultaneous determination of salbutamol sulphate (SS), bromhexine hydrochloride (BH) and etofylline (ET) in pure and commercial formulations without any prior separation or purification. They were first derivative zero crossing spectrophotometry (method 1), simultaneous equation method (method 2), derivative ratio spectra zero crossing method (method 3) and double divisor ratio spectra derivative method (method 4). The ranges for SS, BH and ET were found to be 1-35 μg mL⁻¹, 4-40 μg mL⁻¹ and 5-80 μg mL⁻¹. For methods 1 and 2, the values of limit of detection (LOD) were 0.2314 μg mL⁻¹, 0.4865 μg mL⁻¹ and 0.2766 μg mL⁻¹ and the values of limit of quantitation (LOQ) were 0.7712 μg mL⁻¹, 1.6217 μg mL⁻¹ and 0.9221 μg mL⁻¹ for SS, BH and ET, respectively. For method 3, LOD values were 0.3297 μg mL⁻¹, 0.2784 μg mL⁻¹ and 0.7906 μg mL⁻¹ and LOQ values were 0.9325 μg mL⁻¹, 0.9282 μg mL⁻¹ and 2.6352 μg mL⁻¹ for SS, BH and ET, respectively. For method 4, LOD values were 0.3161 μg mL⁻¹, 0.2495 μg mL⁻¹ and 0.2064 μg mL⁻¹ and LOQ values were 0.9869 μg mL⁻¹, 0.8317 μg mL⁻¹ and 0.6879 μg mL⁻¹ for SS, BH and ET. The precision values were less then 2% R.S.D. for all four methods. The common excipients and additives did not interfere in their determinations. The results obtained by the proposed methods have been statistically compared by means of Student t-test and by the variance ratio F-test.     

Inner filter effect of gold nanoparticles on the fluorescence of quantum dots and its application to biological aminothiols detection

We have demonstrated the design of a new type fluorescence assay based on the inner filter effect (IFE) of gold nanoparticles (AuNPs) on the fluorescence of quantum dots (QDs). With a high extinction coefficient, AuNPs are expected to be capable of functioning as powerful absorbers. QDs with tunable emission wavelength are ideal fluorophores because the emission spectra of the rationally synthesized QDs can perfectly overlap with the absorption band of the absorber. Aminothiols are chosen as the model analytes, and the IFE-based fluorescent method for detection of aminothiols was suggested. Under the optimum conditions, the response is linearly proportional to the concentration of cysteine in the range of 0.05-0.9 μg mL⁻¹. The present IFE-based fluorescent strategy could be also used to detect glutathione and homocysteine. The linear concentration ranges were 0.05-1.0 μg mL⁻¹ for glutathione and 0.01-1.0 μg mL⁻¹ for homocysteine.     

Monolithic silica spin column extraction and simultaneous derivatization of amphetamines and 3,4-methylenedioxyamphetamines in human urine for gas chromatographic-mass spectrometric detection

A simple, sensitive, and specific method with gas chromatography-mass spectrometry was developed for simultaneous extraction and derivatization of amphetamines (APs) and 3,4-methylenedioxyamphetamines (MDAs) in human urine by using a monolithic silica spin column. All the procedures, such as sample loading, washing, and elution were performed by centrifugation. APs and MDAs in urine were adsorbed on the monolithic silica and derivatized with propyl chloroformate in the column. Methamphetamine-d₅ was used as an internal standard. The linear ranges were 0.01-5.0 μg mL⁻¹ for methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) and 0.02-5.0 μg mL⁻¹ for amphetamine (AP) and 3,4-methylenedioxyamphetamine (MDA) (coefficient of correlation ≧0.995). The recovery of APs and MDAs in urine was 84-94%, and the relative standard deviation of the intra- and interday reproducibility for urine samples containing 0.1, 1.0, and 4.0 μg mL⁻¹ of APs and MDAs ranged from 1.4% to 13.6%. The lowest detection limit (signal-to-noise ratio ≧ 3) in urine was 5 ng mL⁻¹ for MA and MDMA and 10 ng mL⁻¹ for AP and MDA. The proposed method can be used to perform simultaneous extraction and derivatization on spin columns that have been loaded with a small quantity of solvent by using centrifugation.     

Fluorescence enhancement of CdTe/CdS quantum dots by coupling of glyphosate and its application for sensitive detection of copper ion

A novel fluorescent probe for Cu²⁺ determination based on the fluorescence quenching of glyphosate (Glyp)-functionalized quantum dots (QDs) was firstly reported. Glyp had been used to modify the surface of QDs to form Glyp-functionalized QDs following the capping of thioglycolic acid on the core–shell CdTe/CdS QDs. Under the optimal conditions, the response was linearly proportional to the concentration of Cu²⁺ between 2.4 × 10⁻² μg mL⁻¹ and 28 μg mL⁻¹, with a detection limit of 1.3 × 10⁻³ μg mL⁻¹ (3δ). The Glyp-functionalized QDs fluorescent probe offers good sensitivity and selectivity for detecting Cu²⁺. The fluorescent probe was successfully used for the determination of Cu²⁺ in environmental samples. The mechanism of reaction was also discussed.     

Sensitive determination of hydrazine in water by gas chromatography–mass spectrometry after derivatization with ortho-phthalaldehyde

A gas chromatography–mass spectrometric (GC–MS) method has been established for the determination of hydrazine in drinking water and surface water. This method is based on the derivatization of hydrazine with ortho-phthalaldehyde (OPA) in water. The following optimum reaction conditions were established: reagent dosage, 40 mg mL⁻¹ of OPA; pH 2; reaction for 20 min at 70 °C. The organic derivative was extracted with methylene chloride and then measured by GC–MS. Under the established condition, the detection and the quantification limits were 0.002 μg L⁻¹ and 0.007 μg L⁻¹ by using 5.0-mL of surface water or drinking water, respectively. The calibration curve showed good linearity with r² = 0.9991 (for working range of 0.05–100 μg L⁻¹) and the accuracy was in a range of 95–106%, and the precision of the assay was less than 13% in water. Hydrazine was detected in a concentration range of 0.05–0.14 μg L⁻¹ in 2 samples of 10 raw drinking water samples and in a concentration range of 0.09–0.55 μg L⁻¹ in 4 samples of 10 treated drinking water samples.     

Determination of andrographolide and dehydroandrographolide in rabbit plasma by on-line solid phase extraction of high-performance liquid chromatography

The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining andrographolide and dehydroandrographolide in rabbit plasma. Plasma samples (100 μL) were injected directly into a C18 SPE column and the biological matrix was washed out for 6 min using 15% aqueous methanol. By rotation of the switching valve, andrographolide and dehydroandrographolide were eluted in the back-flush mode and transferred to the analytical column by the chromatographic mobile phase consisted of methanol:acetonitrile (ACN):water (50:10:40; v/v). The UV detection was performed at 225 nm. The calibration curves showed excellent linear relationship (R ≥ 0.9993) over the concentration range of 0.05-5.0 μg mL⁻¹. The within- and between-day precisions (R.S.D.) of two analytes were in the range of 1.2-6.5% and the accuracies were between 92.0% and 102.1%. Their recoveries were all greater than 94%. The limits of detection were 0.019 μg mL⁻¹ for andrographolide and 0.022 μg mL⁻¹ for dehydroandrographolide. This method was successfully applied to the plasma concentration-time curve study after oral administration of Andrographis paniculata Nees extract in rabbit.     

Sensitive quantitative determination of oxymatrine and matrine in rat plasma by capillary electrophoresis with stacking induced by moving reaction boundary

A quantitative method of capillary electrophoresis with sample stacking induced by moving reaction boundary (MRB) was developed for sensitive determination of oxymatrine (OMT) and matrine (MT) in rat plasma. The experimental conditions were optimized firstly. Below are the optimized experimental conditions: 20 mM sodium formate solution (HCOONa, adjusted to pH 10.70 by ammonia) as sample solution, 3 min 14 mbar sample injection, 40 mM formic buffer (HCOOH-HCOONa, pH 2.60) as stacking buffer, 7 min 14 mbar injection of stacking buffer, 100 mM HCOOH-HCOONa (pH 4.80) as separation buffer, 73 cm capillary (effective length 64 cm), 21 kV voltage, 210 nm wavelength. Under the optimized conditions, higher than 60-fold sensitivity improvement of the stacking was simply achieved as compared with capillary zone electrophoresis, and the detectable limits obtained for OMT and MT were 0.26 and 0.19 μg mL⁻¹, respectively. Then, numerous demonstrations were carefully performed for the methodological validations of OMT and MT in rate plasma, including high specificity of method, good linearity (r = 0.9993 for OMT, r = 0.9991 for MT), fair wide linear concentration range (1.30-65.00 μg mL⁻¹ for OMT, 0.84-42.00 μg mL⁻¹ for MT), low limit of detection (1.03 μg mL⁻¹ for OMT, 0.38 μg mL⁻¹ for MT), less than 5% intra- and inter-day variance value, and higher than 96% recovery of OMT and MT in plasma. The developed method could be used for the trace analyses of OMT and MT in plasma and was finally used for the investigation on pharmacokinetic study of OMT in rat plasma.     

Use of 2-mercaptopyridine for the determination of alkylating agents in complex matrices: application to dimethyl sulfate

A rapid and sensitive method for the determination of alkylating agents in complex reaction mixtures was developed and characterized. Analyses are based on the alkylation of 2-mercaptopyridine by the analyte; the derivative is separated by RP-HPLC and measured by fluorescence detection. When applied to the determination of dimethyl sulfate, the method is linear over four orders of magnitude: 0.01-10 μg mL⁻¹. By using recrystallized 2-mercaptopyridine, quantitation limits of 10 ng mL⁻¹ can be achieved. Precision of the assay is 2% R.S.D. in the 1-10 μg mL⁻¹ range and about 15% R.S.D. at 10 ng mL⁻¹. Studies on the pH dependence of the derivatization reaction were key to minimizing interference from the dimethyl sulfate degradation product, monomethyl sulfate, in quenched reaction samples.     

Determination of mono- and dichloroacetic acids in betaine media by liquid chromatography

A simple and sensitive method has been developed for the analysis of residue amounts of chloroacetic acids in betaine samples based on derivatization by 1-naphthylamine (NA). The derivatized compounds are analyzed by reverse phase high performance liquid chromatography using methanol and water as mobile phase in the ratio of 32/68 (v/v) and phenyl column and PDA detection at 222 nm. The detection limits (LOD) of monochloroacetic acid (MCA) and dichloroacetic acid (DCA) are 0.1 and 0.15 μg mL⁻¹, respectively. The limits of quantification (LOQ) and the linear dynamic ranges (LDR) of MCA are found to be 1 and 1-400 μg mL⁻¹, respectively, and for DCA are found to be 3 and 3-400 μg mL⁻¹, respectively. The precision at the 5 ppm level for MCA and DCA are about 3% and 2%, (n = 5), respectively. The average recovery for MCA and DCA spiked to betaine samples are 98% and 97%, respectively.     

Determination of osthole in rat plasma by high-performance liquid chromatograph using cloud-point extraction

A new straightforward method based on cloud-point extraction (CPE) was developed to determine osthole in rat plasma by reversed phase high-performance liquid chromatography with ultraviolet detection using a photodiode array detector. The non-ionic surfactant Triton X-114 was chosen as the extract solvent. Variable parameters affecting the CPE efficiency were evaluated and optimized. A Zorbax SB-C₁₈ column was used for elution separation at 25 °C with detection wavelength at 322 nm. Under the optimum conditions, the method was shown to be reproducible and reliable with intra-day precision below 7.62%, inter-day precision below 6.37%, and accuracy within ±5.02% and mean extraction recovery more than 90.4%, which were all calculated using a range of spiked samples at three concentrations of 0.5, 5.0 and 15.0 μg mL⁻¹ for osthole in plasma. The calibration curve for the analyte was linear in the range from 0.1 to 20 μg mL⁻¹ with the correlation coefficients greater than 0.9981. Limit of detection (S/N = 3) was less than 0.03 μg mL⁻¹and limit of quantification (S/N = 10) was less than 0.1 μg mL⁻¹. After strict validation, the method was successfully applied to the pharmacokinetic study of osthole in rats after oral and intravenous administration, respectively.     

Determination of azoxystrobin residues in grapes, musts and wines with a multicommuted flow-through optosensor implemented with photochemically induced fluorescence

In this paper, the conversion of azoxystrobin in a strongly fluorescent degradation product by UV irradiation with quantitative purposes and its fluorimetric determination are reported for the first time. A multicommuted flow injection-solid phase spectroscopy (FI-SPS) system combined with photochemically-induced fluorescence (PIF) is developed for the determination of azoxystrobin in grapes, must and wine. Grape samples were homogenized and extracted with methanol and further cleaned-up by solid-phase extraction on C₁₈ silica gel. Wine samples were solid-phase extracted on C₁₈ sorbent using dichloromethane as eluent. Recoveries of azoxystrobin from spiked grapes (0.5-2.0 mg Kg⁻¹), must (0.5-2.0 μg mL⁻¹) and wine (0.5-2.0 μg mL⁻¹) were 84.0-87.6%, 95.5-105.9% and 88.5-111.2%, respectively. The quantification limit for grapes was 0.021 mg Kg⁻¹, being within European Union regulations, and 18 μg L⁻¹ and 8 μg L⁻¹ for must and wine, respectively.     

Dynamic hollow-fibre liquid phase microextraction of dinitrophenols from human plasma: Optimization of an extraction flow system using experimental design methodology

The utility of a dynamic hollow-fibre liquid phase microextraction method (optimized using a four-variable experimental design and response surface modelling) for extracting dinitrophenolic compounds from human plasma samples was evaluated. The investigated variables were donor phase salt concentration (10-400 mM), donor phase pH (2-6), acceptor phase pH (7-12), and donor/acceptor phase flow rates (30/7.5 to 70/17.5 μL min⁻¹). Four dinitrophenol pesticides were used as model substances at concentrations of 0.1 μg mL⁻¹ in spiked human plasma samples. Extraction efficiencies ranging from 42 to 77% with RSDs below 9 were achieved with the optimized method. The flow rate and acceptor pH were shown to strongly affect the extraction efficiency for all compounds, while the donor phase pH and salt concentration had minor effects. With a well-defined acceptor phase pH and flow rate the system exhibited high robustness. The limits of quantification for the investigated compounds, using the presented extraction method followed by liquid chromatography/electrospray ionization mass spectrometry in selected ion monitoring mode, ranged from 0.05 to 0.1 μg mL⁻¹ plasma.     

Derivatized silver nanoparticles as sensor for ultra-trace nitrate determination based on light scattering phenomenon

New silver nanoparticles coated with EDTA (EDTA-AgNPs) have been synthesized by citrate reduction method and characterized by UV-vis spectroscopy, molecular fluorescence and scanning electron microscopy (SEM). The derivatized nanoparticles show fluorescent emission and second order scattering (SOS) signals which in presence of nitrate are both attenuated. The SOS decreasing is greater than its fluorescent quenching; considering this fact, a new ultra sensitive methodology using the derivatized silver nanoparticles as sensor for nitrate determination has been developed. Under optimal established conditions, a linear response has been obtained within the range of 6.4 × 10⁻⁴ to 3.0 μg mL⁻¹ nitrate concentrations, with a detection limit of 1.8 × 10⁻⁴ μg mL⁻¹. This novel technique provides a sensitive and selective methodology for nitrate determination and has been satisfactorily applied to its quantification in parenteral solutions.     

Determination of perfluorocarboxylic acids in water by ion-pair dispersive liquid–liquid microextraction and gas chromatography–tandem mass spectrometry with injection port derivatization

A novel technique for derivatization in a gas chromatograph injection port after a one-step extraction of trace perfluorocarboxylic acids (PFCAs) in water with ion pair formation during dispersive liquid–liquid microextraction (DLLME) was investigated. Tetrabutylammonium hydrogen sulfate (TBAHS) was used as the ion pair reagent. PFCA butyl ester derivatives were formed in the GC injection port and then analyzed using gas chromatography coupled to tandem mass spectrometry with negative chemical ionization. According to our analysis, the operative linear range for PFCA detection from 250 pg mL⁻¹ to 2 μg mL⁻¹ with a relative standard derivation (RSD) below 13%. Detection limits were achieved at the level of 37–51 pg mL⁻¹. This method was successfully applied for the analyzing of PFCAs in river water samples from urban and industrial areas without tedious pretreatment. The concentration range over which PFCAs were detected is from 0.6 ng mL⁻¹ to 604.9 ng mL⁻¹.     

Approaches for application of sub and supercritical fluid extraction for quantification of orbifloxacin from plasma and milk: Application to disposition kinetics

Since its extensive development in the early 1980s, SFE has attracted considerable attention as a sample-preparation procedure. However, other different sample preparation procedures, including precipitation, liquid- and/or solid-phase extraction in biological fluids, also remain in use. In this investigation, SFE was introduced to isolate and identify orbifloxacin from plasma and milk. Four parameters, including the temperature and the pressure of supercritical fluid, modifier ratios, and dynamic extraction time, were evaluated and optimized to obtain the best yield of the analyte from the biological fluids. Determinations of the orbifloxacin (OBFX) in the extracts were carried out using HPLC-FLD. The optimum conditions of the extraction process that yielded the maximum analyte extraction efficiencies were 150 °C vs. 60 °C, 250 kg cm⁻², 30% vs. 35% methanol, and 40 min vs. 20 min, for plasma and milk, respectively. The linearity of the calibration curves as well as the instrument LODs/LOQs were evaluated. Good linearity (at least r² ≥ 0.999) of the calibration curves was obtained over the range from 0.2 to 0.01 μg mL⁻¹. The method showed a good recovery rate (74.2-127.73%) and precision (RSDs: 1.64-20%). The instrumental LOD and LOQ values were 0.004 μg mL⁻¹ vs. 0.01 μg mL⁻¹ or 0.006 μg mL⁻¹ vs. 0.02 μg mL⁻¹, for plasma and milk, respectively. The method was successfully applied to estimate the pharmacokinetic variables of orbifloxacin in lactating does. To the best of our knowledge, this is the first time that SFE has been applied to isolate an antimicrobial agent from biological fluids. This method is promising for clinical applications and for pharmacokinetic studies of various pharmaceuticals in biological fluids.     

Optimization and comparison of chemical and electrochemical hydride generation for optical emission spectrometric determination of arsenic and antimony using a novel miniaturized microwave induced argon plasma exiting the microstrip wafer

Continuous flow (CF) chemical hydride generation (CHG) and electrochemical hydride generation (ECHG) directly coupled to a novel 40 W, atmospheric pressure, 2.45 GHz microwave microstrip Ar plasma exiting a microstrip wafer has been developed for the emission spectrometric determination of As and Sb using a miniaturized optical fiber spectrometer and a CCD-array detector. The experimental conditions for both procedures were optimized with respect to the relative net intensities of the As I 228.8 nm and Sb I 252.8 nm lines and their signal-to-background intensity ratios. Additionally, the susceptibility to interferences from Cd, Co, Cr, Cu, Fe, Ni, Pb and Zn and other hydride-forming elements in the determination of As and Sb using the CHG and ECHG techniques was investigated in detail. Under the optimized conditions, it was found that ECHG is more prone to interferences compared to CHG. The detection limits (3σ) of As (6 ng mL⁻¹) and Sb (7 ng mL⁻¹) obtained for the ECHG-MSP-OES method are about three times lower than in the case of the CHG-MSP-OES method due to a two-fold lower amount of H₂ introduced into the MSP in case of the ECHG, resulting in a better plasma stability and reduced background level. The linearity ranges for both calibration curves to a concentration of up to 5 μg mL⁻¹ and a precision between 2% and 7% (2 μg mL⁻¹ and 0.050 μg mL⁻¹ of As and Sb, respectively) were found for both methods. The developed ECHG-MSP-OES method was validated for As through the analysis of a certified coal fly ash standard reference material (NIST SRM 1633a) after sample dissolution. The derived concentration (140 ± 8 μg g⁻¹) was found to agree well with the certified data (145 ± 15 μg g⁻¹). The method was also successfully applied to the analysis of both a galvanic bath sample, which contained Sb and was spiked with As, and a tap water sample spiked with both analytes. Recovery rates of 99-101% and a Sb concentration of 6.6 μg mL⁻¹ in the galvanic bath sample were revealed. The latter value showed a good agreement with the data obtained from ICP-OES analysis, which was also used for validation purpose.     

Chromogenic platform based on recombinant Drosophila melanogaster acetylcholinesterase for visible unidirectional assay of organophosphate and carbamate insecticide residues

In this study we propose a chromogenic platform for rapid analysis of organophosphate (OP) and carbamate (CM) insecticide residues, based on recombinant Drosophila melanogaster acetylcholinesterase (R-DmAChE) as enzyme and indoxyl acetate as substrate. The visible chromogenic strip had the advantages identical to those of commonly used lateral flow assays (LFAs) with utmost simplicity in sample loading and result observation. After optimization, depending on the color intensity (CI) values, the well-established assay has the capabilities of both qualitative measurement via naked eyes and quantitative analysis by colorimetric reader with the desirable IC₅₀ values against the tested six insecticides (0.06 μg mL⁻¹ of carbofuran, 0.28 μg mL⁻¹ of methomyl, 0.03 μg mL⁻¹ of dichlorvos, 31.6 μg mL⁻¹ of methamidophos, 2.0 μg mL⁻¹ of monocrotophos, 6.3 μg mL⁻¹ of omethoate). Acceptable matrix effects and satisfactory detection performance were confirmed by in-parallel LC–MS/MS analysis in different vegetable varieties at various spiked levels of 10⁻³ to 10¹ μg g⁻¹. Overall, the testified suitability and applicability of this novel platform meet the requirements for practical use in food safety management and environmental monitoring, especially in the developing world.     

Determination of clarithromycin in rat plasma by HPLC-UV method with pre-column derivatization

A novel high-performance liquid chromatographic (HPLC) method using pre-column derivatization and UV detection at 275 nm for the determination of clarithromycin in rat plasma has been validated. Clarithromycin was extracted from plasma sample spiked with internal standard (erythromycin) under alkaline condition with ethyl ether and derivatizated with trimethylbromosilane. The analyses were run on a C₁₈ column, maintained at 40 °C during elution, using a mobile phase comprised of potassium dihydrogen phosphate (50 mM, pH 6.8, contained 0.7% triethylamine), acetonitrile, and methanol (30:45:25, v/v/v). The standard calibration curve for clarithromycin was linear (r² = 0.9998) over the concentration range of 0.1-10 μg ml⁻¹ in rat plasma. The limit of detection (LOD) and limit of quantitation (LOQ) was 30 ng ml⁻¹ and 0.1 μg ml⁻¹ respectively. The intra- and inter-day assay variability range was 2.6-7.4% and 3.3-8.5%, respectively. This method has been successfully applied to a pharmacokinetic study of clarithromycin in rats.