Quantitative analysis of sirolimus (Rapamycin) in blood by high-performance liquid chromatography–electrospray tandem mass spectrometry

We report here a quantitative method for the analysis of sirolimus in blood using solid-phase sample preparation and HPLC–electrospray-tandem mass spectrometry detection. Blood samples (500 μl) were prepared by pre-treatment with acetonitrile: 15 mM zinc sulphate (70:30, v/v), containing 32-demethoxysirolimus (internal standard) and C₁₈ solid-phase extraction. The electrospray conditions were chosen to enhance the [M+NH₄]⁺ species at the expense of other species. Detection was by multiple reactant monitoring with the mass transitions m/z 931.8→864.6 and m/z 901.8→834.4 employed for sirolimus and the internal standard, respectively. The method was linear over the range 0.2 to 100.0 μg l⁻¹. The accuracy and inter-day precision, over this concentration range, was 94.4% to 104.4% and 1.4% to 5.0%, respectively. The accuracy and total precision at the limit of quantitation (0.2 μg l⁻¹) was 103.0% and 10.8%, respectively. The mean absolute recovery of sirolimus and the internal standard were 80.5% and 81.3%, respectively. The sensitivity and analytical concentration range of the method make it suitable for therapeutic drug monitoring and pharmacokinetic studies. Further, the ability of the method to measure parent drug specifically will facilitate the evaluation of immunoassays for sirolimus.     

Automated preparation and analysis of barbiturates in human urine using the combined system of PrepStation and gas chromatography–mass spectrometry

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography–mass spectrometry (GC–MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. Sample clean-up was performed by a solid-phase extraction (SPE) on a C₁₈ disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading a 1.5 ml volume of a urine sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol–water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform–isopropanol (3:1, v/v) from the cartridge. The eluate (1 μl) was injected into a GC–MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 μg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.     

Simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma using high-performance liquid chromatography

A simple, specific and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of rufloxacin, fenbufen and felbinac in human plasma. Plasma, spiked with internal standard, was vortex-mixed for 1 min with a mixture of dichloromethane-diethyl ether (80:20, v/v). The evaporated extract was dissolved in 0.02 M NaOH. Drugs were resolved at room temperature on a 5 μm Zorbax SAX column (250×4.6 min I.D.) equipped with a 20×4.6 mm anion-exchange Vydac AXGU ( 10 μm particle size) precolumn. The mobile phase consisted of acetonitrile and phosphate buffer (pH 7.0), delivered at a flow-rate of 1.2 ml/min. Detection was made at 280 nm, 2-[4-(2′-Furoyl)phenyl]propionic acid was used as internal standard. The calibration curve was linear from 0.2 to 10μg/ml for rufloxacin, from 0.5 to 30 μg/ml for fenbufen and from 0.2 to 10 μg/ml for felbinac, respectively. The detection limit was 0.1 μg/ml for rufloxacin. 0.3 μg/ml for fenbufen and 0.1 μg/ml for felbinac, respectively.     

High-performance liquid chromatographic method for the determination of mycophenolate mofetil in human plasma

A method for the quantification of mycophenolate mofetil (MMF, CellCept) in plasma using solid-phase extraction and HPLC is described here. A solution of internal standard is added to a 0.5-ml plasma aliquot. The resulting sample is treated with water and dilute HCl and applied to a C₁₈ solid-phase extraction column. After a water wash, the MMF and internal standard are eluted with methanol-0.1 M citrate-phosphate buffer, pH 2.6 (80:20, v/v). A 20-μl aliquot of the eluate is injected onto a C₁₈ column (5 μm particle size, 150 × 4.6 mm I.D.) and eluted at ambient temperature with acetonitrile-0.05 M citrate-phosphate buffer, pH 3.6, containing 0.02 M heptanesulfonic acid (41:59, v/v). Quantification is achieved by UV detection at 254 nm. The method is reproducible, accurate and specific for MMF. Using 0.5 ml of plasma for analysis, the quantification limit is 0.400 μg/ml and the range is 0.400–20 μg/ml. Based on the stability profile of MMF in plasma, it is recommended that blood samples collected following intravenous infusion be immediately stored on ice and that plasma be prepared rapidly, immediately stored frozen at −80°C and analyzed within four months of collection.     

Determination of calcium dobesilate in human plasma using ion-pairing extraction and high-performance liquid chromatography

A rapid, simple reversed-phase high-performance liquid chromatographic method with ultraviolet absorbance detection has been developed for the determination of calcium dobesilate in human plasma. Sample processing is based on an ion-pairing extraction with tetra-n-butylammonium hydroxide as cationic pairing ion and dichloromethane. Separation of the investigated calcium dobesilate and 2,4-dihydroxybenzoic acid as internal standard was achieved on a Discovery RP-Amide C₁₆ analytical column with 50 mM, pH 2.5, potassium dihydrogenphosphate buffer–acetonitrile (75:25, v/v) mobile phase. The wavelength was set at 305 nm. The limit of quantitation is 100 ng/ml and the calibration curve is linear up to 50 μg/ml. Within-day and between-day precision expressed as the relative standard deviation is about 10% and the accuracy of the determination did not deviate from 100% by more than ±10%. The developed method was found to be suitable for application in human bioequivalence studies.     

Sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its N-desethyl metabolite in human saliva or cerebrospinal fluid using solid-phase extraction

A sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its primary metabolite in human saliva or cerebrospinal fluid using solid-phase extraction is described. Samples mixed with internal standard and sodium phosphate buffer were applied to an activated C₁₈ solid-phase extraction column. The reconstituted eluate was injected onto a Zorbax RX C₈ column utilizing a mobile phase of 100 mM ammonium acetate (pH 4.0)–isopropyl alcohol–acetonitrile (55:20:25, v/v/v). Fluorescence detection was employed with excitation at 295 nm and emission at 456 nm. Quantitation was achieved using peak-height ratios. The detection response curve was linear from 2 to 850 nM for atevirdine in both human saliva and cerebrospinal fluid and from 2 to 250 nM for the metabolite in human saliva. The method was utilized to analyze cerebrospinal fluid and saliva samples from clinical studies.     

Determination of saquinavir in human plasma by high-performance liquid chromatography

We developed and characterized a high-performance liquid chromatography (HPLC) assay for the determination of saquinavir, an HIV protease inhibitor, in human plasma samples. Extraction of plasma samples with diethyl ether resulted in quantitative recovery of both saquinavir and its stereoisomer Ro 31-8533 which was used as an internal standard. The assay was performed isocratically using 5 mM H₂SO₄ (pH 3.5) and acetonitrile (75.5:24.5, v/v) containing 10 mM tetrabutylammonium hydrogen sulfate (TBA) as a mobile phase, a Nucleosil 3C8 column kept at 45°C and UV detection at 240 nm. Using this method, saquinavir and Ro 31-8533 can be separated from endogenous substances, and in the concentration range of 5–110 ng/ml the relative standard deviations for the determination of saquinavir were below 5%. The detection limit of saquinavir in human plasma was 1 ng/ml. The usefulness of the method was demonstrated by quantification of saquinavir in plasma of human subjects treated with 600 mg of saquinavir per os or 12 mg intravenously.     

Determination of cisapride and norcisapride in human plasma using high-performance liquid chromatography with ultraviolet detection

A simple, rapid and reproducible high-performance liquid chromatographic assay for cisapride and norcisapride in human plasma is described. Samples of plasma (150 μl) were extracted using a C₁₈ solid-phase cartridge. Regenerated tubes were eluted with 1.0 ml of methanol, dried, redissolved in 150 μl of methanol and injected. Chromatography was performed at room temperature by pumping acetonitrile–methanol–0.015 M phosphate buffer pH 2.2–2.3 (680:194:126, v/v/v) at 0.8 ml/min through a C₁₈ reversed-phase column. Cisapride, norcisapride and internal standard were detected by absorbance at 276 nm and were eluted at 4.3, 5.3 and 8.1 min, respectively. Calibration plots in plasma were linear (r>0.998) from 10 to 150 ng/ml. Intraday precisions for cisapride and norcisapride were 3.3% and 5.4%, respectively. Interday precisions for cisapride and norcisapride were 9.6% and 9.0%, respectively. Drugs used which might be coadministered were tested for interference.     

Sensitive assay for measuring amoxicillin in human plasma and middle ear fluid using solid-phase extraction and reversed-phase high-performance liquid chromatography

We developed a sensitive assay to measure amoxicillin in human plasma and midle ear fluid (MEF) using solid-phase extraction and reversed-phase HPLC. Amoxicillin and cefadroxil, the internal standard, were extracted from 50–200 μl of sample with Bond Elut C₁₈ cartridges. The exact was analyzed on a 15 cm × 2 mm, 5μm Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer (pH = 6.5) and 5 mM tetrabutylammonium. The average absolute recovery of amoxicillin and cefadroxil were 91.2 ± 16.6% and 91.0 ± 6.8%, respectively. The limit of quantitation was 0.125 μg/ml with 200 μl sample size. The linear range was from 0.125 to 35.0 μg/ml with correlation coefficients greater than 0.999. These analytic conditions produced a highly sensitive amoxicillin assay in human body fluids without derivatization.     

Enantioselective determination of selfotel in human urine by high-performance liquid chromatography on a chiral stationary phase after derivatization with 9-fluorenylmethyl chloroformate

An analytical method for the enantioselective determination of selfotel in human urine has been developed and validated. The method is based on high-performance liquid chromatography and utilizes CGS 20005 (a selfotel analog) as the internal standard. Urine samples were derivatized in situ with o-phthalic dicarboxaldehyde–3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate (FMOC). Chromatographic separations of the FMOC derivatives of selfotel enantiomers and the internal standard were achieved using a column switching system consisting of an Inertsil ODS-2 column (75×4.6 mm I.D., 5 μm) and a Chiralcel OD-R column (250×4.6 mm I.D., 10 μm). The composition of the mobile phase was acetonitrile–0.1 M phosphate buffer, pH 2.50 (35:65) for the Inertsil ODS-2 column and acetonitrile–0.1 M phosphate buffer, pH 2.00 (35:65) for the Chiralcel OD-R column. The analytes were monitored using fluorescence detection at an excitation wavelength of 262 nm and an emission wavelength of 314 nm. The limit of quantification (LOQ) for this method is 0.25 μg/ml for each selfotel enantiomer. The method was successfully utilized to determine preliminary selfotel stereospecific pharmacokinetics.     

Simultaneous determination of epirubicin, doxorubicin and their principal metabolites in human plasma by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection has been developed for the simultaneous determination of epirubicin, 13-S-dihydroepirubicin, doxorubicin and 13-S-dihydrodoxorubicin in human plasma. An aliquot of 200 μl plasma, spiked with internal standard, was extracted by solid-phase extraction using polymeric adsorbent columns. Chromatography was performed using a C₁₈ reversed-phase column with a mobile phase consisting of water–acetonitrile (71:29, v/v) containing 0.05 M Na₂HPO₄ and 0.05% v/v triethylamine adjusted to pH 4.6 with citric acid. Linearity of the method was obtained in the concentration range of 1–500 ng/ml for all the analytes. Analytical recoveries of the analytes ranged from 89 to 93%. The assay can be used for the simultaneous determination of the four analytes, or for epirubicin and its metabolite or doxorubicin and its metabolite, using the other parent drug as an internal standard. The method was applied to analyze human plasma samples from patients treated with epirubicin using doxorubicin as an internal standard.     

Simultaneous determination of the HIV-protease inhibitors indinavir, amprenavir, ritonavir, saquinavir and nelfinavir in human plasma by reversed-phase high-performance liquid chromatography

A rapid, simple and sensitive high-performance liquid chromatographic (HPLC) assay has been developed for the simultaneous quantification of the HIV-protease inhibitors indinavir, amprenavir, ritonavir, saquinavir and nelfinavir in human plasma. The method involved the solid-phase extraction of the five drugs and the internal standard (I.S., verapamil) from 400 μl of human plasma. The HPLC analysis used a reversed-phase C₁₈ analytical column and a mobile phase consisting of a gradient with 15 mM phosphate buffer (pH 5.75)–acetonitrile and UV monitoring. The method was linear over the therapeutic concentration range for the five HIV-protease inhibitors. The accuracy of the method ranged from 98.2 to 106.7% and the precision values ranged from 1.4 to 8.1% for intra-day precision and from 3.1 to 6.4% for the inter-day values.     

Microscale high-performance liquid chromatography–electrospray tandem mass spectrometry assay for cyclosporin A in blood

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC–electrospray tandem mass spectrometry assay. Blood samples (100 μl) were prepared by acetonitrile precipitation and C₁₈ solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5–1000 μg/l (r≥0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 μg/l and 2.8% at 800 μg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x+10.0, r=0.996). This HPLC–ESI-MS–MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.     

Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether–isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 μl potassium phosphate (0.1 M, pH 2.2) and 60 μl of the acid solution was injected into a C₁₈ BDS Hypersil analytical column (3 μm, 100×4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H₃PO₄)–acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5–100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.     

Determination of the angiotensin-converting enzyme inhibitor lisinopril in urine using solid-phase extraction and reversed-phase high-performance liquid chromatography

A simple, accurate and precise high-performance liquid chromatographic method is described for assaying lisinopril in human urine. Urine (1 ml) containing lisinopril and enalaprilat (internal standard) was acidified with 10 μl of 6 M nitric acid, passed through a Sep-Pak C₁₈ cartridge and eluted with 3 ml of 10% acetonitrile, followed by 6 ml of distilled water. The separations were carried out using a μBondapak C₁₈ column with a mobile phase comprising acetonitrile (60 ml), methanol (10 ml) and tetrahydrofuran (10 ml) in 15 mM phosphate buffer (920 ml) at pH 2.90. Separations were performed at 40°C and detection was at 206 nm. Standard calibration plots of lisinopril in urine were linear (r> 0.998) and recovery was greater than 64%. The lowest quantifiable concentration was 0.5 μg/ml. Within-day and between-day imprecision (coefficient of variation) ranged from 2.51% to 9.26%, and inaccuracy was less than 8.3%.     

Determination of trovafloxacin,a new quinolone antibiotic,in biological samples by reversed-phase high-performance liquid chromatography

A simple, accurate and precise high-performance liquid chromatographic method was developed and validated for the determination of trovafloxacin, a new quinolone antibiotic, in serum and urine. Following solid-phase extraction, chromatographic separation was accomplished using a C₁₈ column with a mobile phase consisting of 0.04 M H₃PO₄-acetonitrile-tetrabutylammonium hydroxide-0.005 M dibutyl amine phosphate (D-4) reagent (83:16.85:0.05:0.1, v/v), pH 3. Trovafloxacin and the internal standard (a methyl derivative of trovafloxacin) were detected by ultraviolet absorbance at 275 nm. The lower limit of quantification for trovafloxacin was 0.1 μg/ml and the calibration curves were linear over a concentration range of 0.1 to 20..0 μg/ml (r² = 0.9997). The average recoveries were greater than 70% for both trovafloxacin and internal standard. The intra-day and inter-day coefficients of variation were generally less than 5% in urine and serum over the concentration range of 0.1 to 20.0 μg/ml. Human serum samples could be stored for up to 12 months at −20°C and urine samples could be stored up to 18 months at −80°C.     

Determination of nicotine and cotinine in human plasma by liquid chromatography-tandem mass spectrometry with atmospheric-pressure chemical ionization interface

Here we report a sensitive liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method capable of quantifying nicotine down to 1 ng/ml and cotinine to 10 ng/ml from 1.0 ml of human plasma. The method was validated over linear ranges of 1.0–50.0 ng/ml for nicotine and 10.0–500.0 ng/ml for cotinine, using deuterated internal standards. Compounds were simply extracted from alkalinized human heparinized plasma with methylene chloride, reconstituted into a solution of acetonitrile, methanol and 10 mM ammonium acetate (53:32:15, v/v) after the organic phase was dried down, and analyzed on the LC-MS-MS, which is a PE Sciex API III system equipped with a Keystone BDS Hypersil C₁₈ column and atmospheric pressure chemical ionization (APCI) interface. The between-run precision and accuracy of the calibration standards were ≤6.42% relative standard deviation (R.S.D.) and ≤11.8%n relative error (R.E.) for both nicotine and cotinine. The between-run and within-run precision and accuracy of quality controls. (2.5, 15.0, 37.5 ng/ml for nicotine and 25.0, 150.0, 375.0 ng/ml for cotinine), were ≤6.34% R.S.D. and ≤7.62% R.E. for both analytes. Sample stabilities in chromatography, in processing and in biological matrix were also investigated. This method has been applied to pharmacokinetic analysis of nicotine and cotinine in human plasma.     

Column liquid chromatographic determination of paroxetine in human serum using solid-phase extraction

A 0.5-ml aliquot of a serum sample, after the addition of a 100-μl aliquot of a 5 μg/ml solution of dibucaine as the internal standard, is vortex-mixed with 0.5 ml of acetonitrile and centrifuged. The supernatant is applied to a 1-ml BondElut C₁₈ silica extraction column conditioned with subsequent washings with 1 M HCl, methanol and water. After passing the sample at a slow rate, the column is washed twice with water and once with acetonitrile. The desired compounds are then eluted with a 0.25-ml aliquot of 35% perchloric acid—methanol (1:40, v/v). A 7-μl aliquot of the eluate is injected onto a 150 × 4.6 mm I.D. column packed with 5-μm C₈ silica particles and eluted at ambient temperature with a mobile phase of 10 mM phosphate buffer-acetonitrile (2:1, v/v) (pH 3.2). The peaks are detected with a fluorescence detector (excitation at 295 nm, emission at 365 nm). The resulting chromatogram is clean with no extraneous peaks. Paroxetine and dibucaine give sharp peaks which are well separated from each other and from the solvent peaks. The extraction recovery of the drug and the internal standard is in the range of 90% which allows a highly sensitive determination of paroxetine.     

Determination of a new fluoroquinolone antimicrobial agent, (S)-10-[(S)-(8-amino-6-azaspiro[3,4]octan-6-yl]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-de[1,4] benzoxazine-6-carboxylic acid hemihydrate,DV-7751a,in human serum and urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of a new fluoroquinolone antimicrobial agent, (S)-10-[(S)-(8-amino-6-azaspiro[3,4]octan-6-yl)]-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido [1,2,3-de][1,4]benzoxazine-6-carboxylic acid hemihydrate (DV-7751a, I) in human serum and urine has been developed. Compound I and the internal standard were extracted from serum and urine by means of Bond Elut C₈ LRC column. The extracts were chromatographed on a reversed-phase Inertsil ODS-2 column using tetrahydrofuran-50 mM KH₂PO₄ (pH 2)-1 M ammonium acetate (19:81:1, v/v) as the mobile phase at a flow-rate of 1.0 ml/min. Fluorescence detection at an excitation wavelength of 305 nm and an emission wavelength of 530 nm resulted in a limit of quantitation of 0.0098 μg/ml for serum and 0.098 μg/ml for urine. The method showed satisfactory sensitivity, precision, accuracy, recovery and selectivity. Stability studies showed that I was stable in serum and urine for at least 1 month at −20°C and for at least 48 h at room temperature.