High-performance Liquid Chromatographic Analysis of Pioglitazone,Gliquidone,Rosuvastatin and Simvastatin in Formulations and Human Serum

Co‐administration of HMG‐CoA reductase with antidiabetic drugs is most common since antidiabetic drugs are mostly prescribed for long term therapy. In the present paper, we describe the simultaneous determination of antidiabetic (pioglitazone hydrochloride and gliquidone) in presence of statins (rosvastatin and simvastatin) in formulations and in human serum using RP‐HPLC technique. The serum samples were subjected to protein precipitation with acetonitrile prior to an HPLC analysis. At a flow rate of 1 mL·min⁻¹ isocratic elution was employed, using mobile phase consisting of methanol/water (90:10, V:V), pH 3.50 with phosphoric acid and absorbance was recorded at 235 nm. The assay was reproducible, linear (concentration range of 5–50 μg·mL⁻¹) and accurate. The LOD and LOQ values were 1.32, 0.28, 0.05 and 0.57 μg·mL⁻¹ and 4.39, 0.93, 0.16 and 1.90 μg·mL⁻¹ for pioglitazone hydrochloride, gliquidone, rosvastatin and simvastatin, respectively. There were no interfering peaks due to the excipients present in the pharmaceutical tablet and serum. Thus, the proposed method is simple and suitable for the analysis of active ingredient in tablet form and human serum.     

Simultaneous Quantitation of Captopril and NSAID's in API,Dosage Formulations and Human Serum by RP‐HPLC

An accurate, sensitive and least time consuming reverse phase high performance liquid chromatographic (RP‐HPLC) method for the estimation of captopril in the presence of non steroidal anti‐inflammatory drugs in formulation and human serum has been developed and validated. Chromatographic separation was conducted on prepacked Purospher star C18 (5 μm, 25 × 0.46 cm) column at room temperature using methanol:water (80:20 v/v) as a mobile phase, pH adjusted at 2.8 with o‐phosphoric acid and at a flow rate of 1.0 mL min⁻¹, while UV detection was performed at 227 nm. The limit of detection and quantification for captopril were 1 and 0.35 ng mL⁻¹, while that for (NSAID's) i.e. flurbiprofen, ibuprofen, diclofenac sodium and mefenamic acid LOD were 0.2, 1, 2 and 0.4 ng mL⁻¹ respectively and LOQ were 0.9, 2.9, 8 and 1 ng mL⁻¹ Analytical recovery was > 98.1%. The method used for the quantitative analysis of commonly administered non steroidal anti‐inflammatory drugs (NSAID's) i.e. ibuprofen, flurbiprofen, diclofenac sodium and mefenamic acid alone or in combination with captopril from API (active pharmaceutical ingredients), dosage formulations and in human serum. The established method is rapid (RT < 12 min), accurate (recovery > 98.1%), selective (no interference of excepients and other commonly used drugs and food) and sensitive (LOQ 3.5 ng mL^;‐1) and reproducible (SD ± 0.003).     

Development of a Rapid and Simultaneous Detection Method for Buprenorphine,Norbuprenorphine and Naloxone in Human Plasma Using Ultra‐high Performance Liquid Chromatography‐tandem Mass Spectrometer with Solid‐phase Extraction

A simultaneous method was successfully established and validated for the separation and determination of buprenorphine (BP), its primary metabolite, nor‐buprenorphine (NBP) and a proposed co‐formulate, naloxone (NLX) in human plasma. The method used buprenorphine‐d₄ (BP‐D₄), nor‐buprenorphine‐d₃ (NBP‐D₃), naltrexone (NTX) as internal standards (ISs). 100 μL of plasma sample fortified with the ISs was cleaned up by solid‐phase extraction (SPE), and was then separated on a Waters Acquity^TM BEH C₁₈ column with gradient elution using methanol and water (containing 0.2% formic) at a flow rate of 0.25 mL·min⁻¹. The mass spectrometer was used for detection and was operated in the positive electrospray ionization with multiple reaction monitoring (MRM) mode. The three compounds were effectively separated in 5 min. The linear ranges of the compounds were 0.1–25, 0.25–25 and 0.05–25 ng·mL⁻¹ for BP, NBP and NLX, respectively, with r≧0.9935. The method had high sensitivity (the limits of detection were 0.02, 0.1 and 0.01 ng·mL⁻¹ for BP, NBP and NLX, respectively) and high recoveries (≧97.6%). The result was shown to be linear and satisfactorily met current acceptance criteria for validation of bioanalytical method: intra and inter assay precisions within the required limits of ≦25% RSD. The LOQs fulfilled the LOQ requirements: precision≦25% RSD, and was fully validated according to the State Food and Drug Administration (SFDA) regulations. The results demonstrated that ultra‐high performance liquid chromatography‐tandem mass spectrometer (UPLC‐MS/MS) with SPE was a powerful detection tool and contributed to pharmaceutical analysis in biological matrices.     

Visible Light Photoelectrochemical Sensor for Acetaminophen Determination using a Glassy Carbon Electrode Modified with BiVO4 Nanoparticles

A new and simple photoelectrochemical (PEC) sensor using a glassy carbon electrode (GCE) modified with bismuth vanadate (BiVO₄) nanoparticles and dihexadecyl phosphate (DHP) film was useful for acetaminophen (AC) determination. In 0.2 mol L⁻¹ phosphate buffer (pH=9), the GCE without modification exhibited the smaller photocurrent (0.86 μA) when compared with GCE modified with 1.0 mg mL⁻¹ or 2.0 mg mL⁻¹ BiVO₄ nanoparticles suspension (5.9 and 34 μA, respectively). Based on the photocurrent signal generated through the interaction between GCE, BiVO₄ and the energy of visible light a chronoamperometric method for AC determination was developed. The AC linear range concentration from 0.099 to 0.99 μmol L⁻¹ and limits of detection and quantification of 0.027 and 0.091 μmol L⁻¹, respectively, was obtained. The proposed method was applied to the AC determination in commercial drugs and tap water with satisfactory accuracy and precision. Moreover, the PEC construction was easy and had a short response time, which might confer higher sample throughput for the method.     

Drug screening in human plasma by cloud-point extraction and HPLC

Cloud-point extraction (CPE) with RP-HPLC/DAD detection was used to develop a screen for six model basic drugs (paracetamol, promazine, amitriptyline, nortriptyline, clomipramine and chlorpromazine) in human plasma. These drugs’ varied hydrophobicities entail different affinities for the micelle-rich phase and CPE extraction efficiencies. Extraction recovery (except paracetamol) was above 80% and reproducibility (RSD%) ranged from 2.88 to 10.26 intraday and from 3.12 to 12.33 interday. The limits of detection were: 0.125 μg mL^?1 (promazine and chlorpromazine), 0.25 μg mL^?1 (amitriptyline and nortriptyline) and 0.5 μg mL^?1 (paracetamol and clomipramine). The method was linear over the ranges: 0.125–1.0 μg mL^?1 (promazine and chlorpromazine), 0.25–1.0 μg mL^?1 (amitriptyline and nortriptyline), 0.5–1.0 μg mL^?1 (clomipramine) and 0.5–10 μg mL^?1 (paracetamol). The procedure is a good alternative to the SPE or LLE sample preparation usually used.     

Analysis of Bile Acids Profile in Human Serum by Ultrafiltration Clean-up and LC-MS/MS

Bile acids (BAs) are useful biomarkers for the diagnosis of many diseases. The pathologies related to bile acid synthesis are often expressed in the first years of life and may lead to serious liver injury. Here we present a sensitive and rapid method for the analysis of the main 14 bile acids in human serum by liquid chromatography-tandem mass spectrometry. The chromatographic separation is performed using a core–shell column which provides improved separation, highly desirable considering the small structural differences among the analytes. All isomeric BAs of interest were resolved in less than 9 min. Sample pretreatment consisted in ultrafiltration of serum after addition of methanol by means of centrifugal filter devices. The calculated LOQs ranged between 2 and 5 ng mL⁻¹ with linearity of the calibration curves in the 5–5,000 ng mL⁻¹ range for all the BAs. The extraction recoveries for all the analytes were higher than 80 %. Intra-day and inter-day coefficients of variation were all below 15 %. The method proposed has been validated and has been applied for the analysis of serum of pediatric patients. This simple procedure allowed minimal consumption of serum sample (about 100 μL) and a rapid assay, easily implementable in routine analysis.

     

Voltammetric Method for the Simultaneous Determination of Melatonin and Pyridoxine in Dietary Supplements Using a Cathodically Pretreated Boron‐doped Diamond Electrode

The simultaneous voltammetric determination of melatonin (MT) and pyridoxine (PY) has been carried out at a cathodically pretreated boron‐doped diamond electrode. By using cyclic voltammetry, a separation of the oxidation peak potentials of both compounds present in mixture was about 0.47 V in Britton‐Robinson buffer, pH 2. The results obtained by square‐wave voltammetry allowed a method to be developed for determination of MT and PY simultaneously in the ranges 1–100 μg mL⁻¹ (4.3×10⁻⁶–4.3×10⁻⁴ mol L⁻¹) and 10–175 μg mL⁻¹ (4.9×10⁻⁵–8.5×10⁻⁴ mol L⁻¹), with detection limits of 0.14 μg mL⁻¹ (6.0×10⁻⁷ mol L⁻¹) and 1.35 μg mL⁻¹ (6.6×10⁻⁶ mol L⁻¹), respectively. The proposed method was successfully to the dietary supplements samples containing these compounds for health‐caring purposes.     

Spectrophotometric Determination of Selected Antibiotics Using Prussian Blue Reaction

A simple, sensitive and accurate spectrophotometric method has been described for the determination of ampicillin(I), amoxicillin trihydrate(II) and cefazolin sodium(III). The procedure is based on the formation of Prussian Blue (PB) complex. The reaction between the acidic hydrolysis products of antibiotics (T = 60 °C) with mixture of Fe³⁺ and hexacyanoferrate(III) ions was evaluated for the spectrophotometric determination of the mentioned drugs. The maximum absorbance of the colored complex occurs at λ = 700 nm and the molar absorptivity is 3.0 × 10⁴ 1 mol^?1cm^?1. The effect of various parameters such as concentration of K₃Fe(CN)₆ and Fe³⁺, nature and amount of acids used, temperature and time of heating were investigated. Under optimum conditions the linear range of calibration graph was 2.0–12.0, 5.0–13.5 and 3.0–12.0 μg mL^?1 for ampicillin, amoxicillin and cefazolin, respectively. The relative standard deviation for the determination of 10 μg mL^?1 of antibiotics was about 0.5–1.5%. The proposed method was successfully applied to the determination of selected antibiotics from pharmaceutical preparations. The validity of the method was tested by the official methods and by the recovery studies of standard addition to pharmaceuticals.     

An LC–MS–MS Method for the Simultaneous Quantitation of Acyclovir and Valacyclovir in Human Plasma

A simple, sensitive and selective LC–MS–MS method has been developed for the simultaneous determination of acyclovir and valacyclovir in human plasma. Acyclovir and valacyclovir in plasma were concentrated by solid phase extraction and chromatographed on a C18 column using a mobile phase of 0.1% formic acid: methanol (30:70% v/v). The method was validated over a linear range of 47–10,255 and 5–1,075 ng mL^?1 for acyclovir and valacyclovir respectively. The LOQs were 47.6 and 5.0 ng mL^?1. The validated method was applied for the quantitation of acyclovir and valacyclovir from plasma samples in a pharmacokinetic study.     

Simple determination of betaine,l‐carnitine and choline in human urine using self‐packed column and column‐switching ion chromatography with nonsuppressed conductivity detection

A sequential online extraction, clean‐up and separation system for the determination of betaine, l ‐carnitine and choline in human urine using column‐switching ion chromatography with nonsuppressed conductivity detection was developed in this work. A self‐packed pretreatment column (50 × 4.6 mm, i.d.) was used for the extraction and clean‐up of betaine, l ‐carnitine and choline. The separation was achieved using self‐packed cationic exchange column (150 × 4.6 mm, i.d.), followed by nonsuppressed conductivity detection. Under optimized experimental conditions, the developed method presented good analytical performance, with excellent linearity in the range of 0.60–100 μg mL⁻¹ for betaine, 0.75–100 μg mL⁻¹ for l ‐carnitine and 0.50–100 μg mL⁻¹ for choline, with all correlation coefficients (R²) >0.99 in urine. The limits of detection were 0.15 μg mL⁻¹ for betaine, 0.20 μg mL⁻¹ for l ‐carnitine and 0.09 μg mL⁻¹ for choline. The intra‐ and inter‐day accuracy and precision for all quality controls were within ±10.32 and ±9.05%, respectively. Satisfactory recovery was observed between 92.8 and 102.0%. The validated method was successfully applied to the detection of urinary samples from 10 healthy people. The values detected in human urine using the proposed method showed good agreement with the measurement reported previously.     

Voltammetric Analysis of Cadmium and Lead (Undesirable Elements) in Brine,Supported by Solid Phase Extraction – the Right Solution of the Analytical Challenge

The stripping voltammetry at HMDE is proposed for Cd and Pb (undesirable ingredients) determination in the natural brine (C_Cl >43 g L⁻¹). Samples with so high salinity have to be significantly diluted. For ICP MS, a 10^5–6 times dilution is required, which disqualifies this method. The proposed procedure allows to determine Cd (0.001 μg L⁻¹) and Pb (0.005 μg L⁻¹) after only 100 times dilution. The thermal chloride stripping or isolation by Chelex 100 increase the quality of obtained data. The recovery study was performed. The LOQs are below recommendations related to the use of brines in balneology.     

On‐line preconcentration strategy for the simultaneous quantification of three local anesthetics in human urine using CZE

The simultaneous determination of usually employed anesthetics (procaine, lidocaine, and bupivacaine) has been developed and validated using CE with ultraviolet detection at 212 nm. The separation of these three drugs has been achieved in less than 7 min, using a temperature of 25ºC and 25 kV, with a 150 mM citrate buffer (pH 2.5) as BGE. Field‐amplified sample injection (FASI) has been used for on‐line sample preconcentration. Ultrapure water and ACN 50/50 (v/v) mixture gave the greatest enhancement factor when it was employed as an injection solvent. Injection voltage and time were optimized, being 13 kV and 13 s, the optimum values, respectively. To avoid the possible irreproducibility associated with the electrokinetic injection, an internal standard such as tetracaine, was employed. The instrumental detection limits (LOD S/N = 3) for the compounds ranged between 2.6 and 7.0 μg L⁻¹ and the quantitation limits (LOQ S/N = 10) between 37.8 and 55.9 μg L⁻¹. The detection limits obtained in real human urine samples ranged between 55.2 and 83.6 μg L⁻¹ and the quantitation limits between 196.0 and 276.0 μg L⁻¹. The proposed method has demonstrated its applicability to the analysis of these local anesthetics in urine samples without any pretreatment, allowing the rapid determination of these target analytes.     

The Application and Validation of HybridSPE-Precipitation Cartridge Technology for the Rapid Clean-up of Serum Matrices (from Phospholipids) for the Clinical Analysis of Serotonin,Dopamine and Melatonin

Phospholipids have been shown to cause matrix effects particularly in liquid chromatography–mass spectrometry (LC–MS) analysis of small molecules. This results in suppression of the analyte signal. This study provides a versatile validated method for the analysis of serotonin in serum along with dopamine and melatonin using LC–MS/MS. It utilises HybridSPE-Precipitation cartridges for the clean-up of serum samples. This technology involves a simple protein precipitation step together with a fast and robust SPE method that is designed to remove phospholipids. Serotonin and dopamine are major neurotransmitters in the brain which affect various functions both in the brain and in the rest of the body. Melatonin plays an important role in the regulation of circadian sleep–wake cycle. Good linear calibrations were obtained for the multiplex assay of analytes in serum samples (0.021–3.268 μmol L⁻¹; R ² = 0.9983–0.9993). Acceptable intra- and inter-day repeatability was achieved for all analytes in serum. Excellent limits of detection (LOD) and limits of quantitation (LOQ) were achieved with LODs of 3.2–23.5 nmol L⁻¹ and the LOQs of 15.4–70.5 nmol L⁻¹ for these analytes in serum. The sample clean-up procedure that was developed provided efficient recovery and reproducibility while also decreasing preparation time and solvent use. A sample storage protocol was established, this was achieved by investigation of sample stability under different storage conditions. Evaluation of matrix effects was also carried out and the influence of ion suppression on analytical results reported. This clean-up protocol was then applied to the analysis of clinical serum samples.

     

Simultaneous determination of synthetic edible pigments in beverages by titania-based RP-HPLC

A rapid method for simultaneous determination of five synthetic edible pigments (SEPs) including tartrazine (TA), ponceau 4R (PO), sunset yellow (SY), brilliant blue (BB) and erythrosine (ER) in beverages with titania-based RP-HPLC has been developed. The good linear relationships were obtained in the concentration ranges of 2.5–40 μg mL⁻¹ for TA, PO and SY, 0.75–12 μg mL⁻¹ for BB, and 1.25–20 μg mL⁻¹ for ER, respectively. The detection limits (LODs) of five SEPs were 0.042, 0.021, 0.042, 0.0005, and 0.021 μg mL⁻¹, respectively. The average recoveries were between 92.2% and 106.3%. Relative standard deviations (RSD, n = 7) for five SEPs were less than 1.18%. The precision and accuracy of the method can meet the requirements of HPLC analysis. In addition, the thermodynamic parameters of retention of the pigments in the titania column such as enthalpy (ΔH^ο), entropy (ΔS^ο) and Gibbs free energy (ΔG^ο) were also determined.     

HPLC and UPLC Methods for the Simultaneous Determination of Enalapril and Hydrochlorothiazide in Pharmaceutical Dosage Forms

High efficiency and less elution are the basic requirements of high-speed chromatographic separation. In this study, a new gradient reverse phase chromatographic methods were developed using HPLC and UPLC systems for simultaneous determination of enalapril maleate (ENL) and hydrochlorothiazide (HCZ) in pharmaceutical dosage forms. The chromatographic separations of ENL and HCZ were achieved on a Waters μ-Bondapak C 18, (300 × 3.9 mm, 10 μm) and Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm) columns for HPLC within 5.30 min and UPLC within a short retention time of 1.95 min, respectively. A linear response was observed over the concentration range 0.270–399 μg mL⁻¹ of ENL, 0.260–399 μg mL⁻¹ of HCZ for HPLC system and 0.270–399 μg mL⁻¹ of ENL and 0.065–249 μg mL⁻¹ of HCZ for UPLC system. Also, limit of detection for ENL was 1.848 ng mL⁻¹ and 31.477 ng mL⁻¹ for HCZ, 2.804 ng mL⁻¹ for ENL and 2.943 ng mL⁻¹ for HCZ using HPLC and UPLC, respectively. The proposed methods were validated according to ICH guideline with respect to precision, accuracy, and linearity. Forced degradation studies were also performed for both compounds in bulk drug samples to demonstrate the specificity and stability indicating power of the HPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency, and resolution.

     

Simultaneous determination and pharmacokinetic study of giraldoid a,giraldoid B in rat plasma after oral administration of Daphne giraldii Nitsche extracts by LC–MS/MS

A simple sensitive LC–MS/MS method has been developed for the simultaneous determination of giraldoid A and giraldoid B in rat plasma. The method was applied to pharmacokinetics studies of the two compounds from Daphne giraldii Nitsche. Chromatographic separation was accomplished on an Acquity UPLC™ BEH C₁₈ column (100 × 2.1 mm, 1.7 mm) by gradient elution with a flow rate of 0.2 mL min⁻¹_. The method was linear over the concentration range of 1.0–1000 ng mL⁻¹, and the lower limits of quantification were 1.04 ± 0.10 and 1.04 ± 0.09 ng mL⁻¹, respectively. The intra‐ and inter‐day precisions (RSD) were <10.14 and 9.96%. The extraction recovery of the analytes was acceptable. Stability studies demonstrated that the two compounds were stable in the preparation and analytical process. The maximum plasma concentration was 687.78 ± 243.62 ng mL⁻¹ for giraldoid A and 952.38 ± 131.99 ng mL⁻¹ for giraldoid B. The time to reach the maximum plasma concentration was 0.50 ± 0.37 h for giraldoid A and 0.50 ± 0.66 h for giraldoid B. The validated method was successfully applied to investigate the concentration–time profiles of giraldoid A and giraldoid B.     

A New Spectrophotometric Method for the Determination of Ascorbic Acid Using Leuco Malachite Green

A new simple and sensitive and selective spectrophotometric method has been developed for the determination of ascorbic acid (AA) at trace level using a new reagent, leuco malachite green (LMG). AAreacts with potassium iodide‐iodate solution under acidic conditions to liberate iodine and the liberated iodine selectively oxidizes LMG to MG dye. The colour of the dye was measured at 620 nm. Beer's law is obeyed over the concentration range of 0.8–8 iμg μAA per 25 mL of final solution (0.032–0.32 ppm). The apparent molar absorptivity and Sandell's sensitivity of the method were found to be 2.98 × 10⁵ l mol⁻¹ cm⁻¹, 0.0042 μg cm⁻², and respectively. Statistical treatment of the experimental results indicates that the method is precise and accurate. The method is free from interference of common ions and many of the ingredients commonly found in pharmaceuticals. The reliability of the method was established by parallel determination against Leucocrystal violet (LCV) method. The method described was satisfactorily applied for the determination of AA in fruit juices, pharmaceuticals and biological samples.     

Differential Pulse Voltammetric Simultaneous Determination of Four Anti‐Inflammatory Drugs by Using Soft Modelling

An application of a partial least squares calibration method for the simultaneous voltammetric determination of indomethacin, acemethacin, piroxicam and tenoxicam is suggested. It was shown that it is possible to resolve complex mixtures of analytes even when they have strongly overlapped signals. In order to check the proposed method, statistical analysis of the results was performed by mean of hypothesis tests. The method developed was applied to the electrochemical reduction region of four anti‐inflammatory drugs and allowed the drugs to be quantified at concentrations between 0.52 and 4.09 μg mL^?1 for acemethacin, 0.44 and 3.50 μg mL^?1 for indomethacin, 0.43 and 3.40 μg mL^?1 for piroxicam, and 0.42 and 3.30 μg mL^?1 for tenoxicam with good results. The average absolute value of relative errors was 2.25%, 4.31%, 1.68% and 2.49%, respectively.     

Quantitative determination of clenbuterol, salbutamol and tulobuterol enantiomers by capillary electrophoresis

Enantiomers of clenbuterol, salbutamol and tulobuterol were directly separated and quantitated from a spiked sample by capillary electrophoresis (CE) using sulfated β-cyclodextrin (SCD) as chiral selector and phosphate as running buffer. The SCD and buffer concentration, pH and field strength were the parameters studied to optimize the separation. Optimal separation was obtained using 50 mM of phosphate monobasic at pH = 2.24, 0.25% (w/w) of sulfated cyclodextrin and a field strength of 10 kV, with 20 min total time analysis. Comparison between two different injection modes (hydrodynamic and electrokinetic) was made. In the hydrodynamic mode, repeatability (expressed as relative standard deviation, RSD) was less than 1.2% for migration times for all the analyte peaks and less than 2% for peak area percentages. With respect to reproducibility, RSD was less than 3.8% for migration time and less than 3% for peak area percentages. Calibration curves were set up for two different sample concentration ranges (1 to 10 μg mL^–1 and 160– 800 ng mL^–1, of each of the racemates studied). Although the electrokinetic injection mode for an aqueous sample appeared to suffer from some enantiodiscrimination, calibration curves were linear in the range between 1 and 10 ng mL^–1 with regression coefficients ranging from 0.9996 to 0.9952. As in the case of hydrodynamic injection, the method was tested with a spiked sample.     

A Simple and Sensitive LC–MS/MS Method for Simultaneous Determination of Temsirolimus and Its Major Metabolite in Human Whole Blood

A simple, fast and sensitive LC–MS/MS method was developed and validated for the simultaneous determination of the concentrations of temsirolimus and its major metabolite, sirolimus, in human whole blood. The blood sample (100 μL) after adding temsirolimus-d7 and sirolimus-d3 internal standards was precipitated with 0.200 mL of methanol/0.300 M zinc sulfate (70/30, v/v), then analyzed by a Shimatzu LC system coupled to a Sciex API-5000 mass spectrometer. The chromatographic separation was carried out on a BDS Hypersil C8 column (50 × 3.0 mm, 5 μm) at 50 °C with a mobile phase composed of methanol/water/formic acid (72/28/0.1) (v/v/v) containing 2.50 mM ammonium acetate. Mass spectrometric detection was performed using electrospray positive ionization with multiple reaction monitoring mode. This method was validated from 0.250 to 100 ng mL⁻¹ for temsirolimus and 0.100 to 40.0 ng mL⁻¹ for sirolimus. The lower limits of quantitation were 0.25 ng mL⁻¹ for temsirolimus and 0.1 ng mL⁻¹ for sirolimus. The intra-day and inter-day precisions (CV %) of spiked quality control (QC) samples were less than 10.4 and 9.6 %, respectively. The accuracies as determined by the relative error for QC samples were less than 12.1 % for intra-day and 7.3 % for inter-day. No significant matrix effect was observed. This method has been successfully applied to analyze clinical pharmacokinetic study samples. The assay reproducibility was also demonstrated by using incurred samples.