Development and validation of a cost-effective and sensitive bioanalytical HPLC-UV method for determination of lopinavir in rat and human plasma

A simple, sensitive and cost-effective HPLC-UV bioanalytical method for determination of lopinavir (LPV) in rat and human plasma was developed and validated. The plasma sample preparation procedure includes a combination of protein precipitation using cold acetonitrile and liquid–liquid extraction with n-hexane–ethyl acetate (7:3, v/v). A good chromatographic separation was achieved with a Phenomenex Gemini column (C₁₈, 150 mm × 2.0 mm, 5 μm) at 40°C with gradient elution, at 211 nm. Calibration curves were linear in the range 10–10,000 ng/mL, with a lower limit of quantification of 10 ng/mL using 100 μL of plasma. The accuracy and precision in all validation experiments were within the criteria range set by the guidelines of the Food and Drug Administration. This method was successfully applied to a preliminary pharmacokinetic study in rats following an intravenous bolus administration of LPV. Moreover, the method was subsequently fully validated for human plasma, allowing its use in therapeutic drug monitoring (TDM). In conclusion, this novel, simple and cost-efficient bioanalytical method for determination of LPV is useful for pharmacokinetic and drug delivery studies in rats, as well as TDM in human patients.     

Development and validation of a high-performance liquid chromatography-electrospray ionization-mass spectrometry assay for the determination of zaleplon in human plasma

In this paper, a sensitive and rapid chromatographic procedure using a selective analytical detection method (electrospray ionization-mass spectrometry in selected-ion monitoring mode) in combination with a simple and efficient sample preparation step is first presented for the determination of zaleplon in human plasma. The separation of the analyte, internal standard, and possible endogenous compounds are accomplished on a phenomenex Luna 5-microm C8(2) column (250- x 4.6-mm i.d.) with methanol-water (75:25, v/v) as the mobile phase. In order to optimize the mass detection of zaleplon, several parameters such as ionization mode, fragmentor voltage, m/z ratios of ions monitored, type of organic modifier, and eluent additive in the mobile phase are discussed. An internal standard is selected to guarantee the quantitative accuracy. Each analysis takes less than 6 min. The calibration curve of zaleplon in the range of 0.1-60.0 ng/mL in plasma is linear with a correlation coefficient of > 0.9992, and the detection limit (s/n = 3) is 0.1 ng/mL. The within- and between-day variations (relative standard deviation) in the zaleplon plasma analysis are less than 2.4% (n = 15) and 4.7% (n = 15), respectively. The application of this method is demonstrated for the analysis of zeleplon plasma samples in a Phase-I human pharmacokinetic study.     

Development and validation of a liquid chromatographic-mass spectrometric assay for the determination of sumatriptan in plasma

Sumatriptan succinate is a novel compound currently in development for the acute treatment of migraine. During early studies in man a sensitive and selective assay was required, which had to be developed rapidly, to determine plasma concentrations following an intravenous infusion. Thermospray liquid chromatography-mass spectrometry combined with the advanced automated sample processor was selected to achieve this. Although the assay was required quickly criteria for intra- and inter-assay accuracies and precisions of +/- 10% had to be achieved. These were obtained only by using a co-eluting deuterium-labelled internal standard. Attempts to use a homologue as an internal standard, which did not co-elute with sumatriptan, gave inferior results. The assay was linear over the calibration range 2-50 ng/ml with a limit of quantification of 2 ng/ml. The application of the technique to the analysis of samples from a volunteer study is demonstrated.     

Development and validation of an HPLC-UV method for the simultaneous quantification of carbamazepine, oxcarbazepine, eslicarbazepine acetate and their main metabolites in human plasma

For the first time, a simple, selective and accurate high-performance liquid chromatography method with ultraviolet detection was developed and validated to quantify simultaneously three structurally related antiepileptic drugs; carbamazepine, oxcarbazepine, and the recently launched eslicarbazepine acetate and their main metabolites, carbamazepine-10,11-epoxide, 10,11-trans-dihydroxy-10,11-dihydro-carbamazepine, and licarbazepine. The method involves a solid-phase extraction and a reverse-phase C18 column with 5 cm length. The mobile phase consisting of water, methanol, and acetonitrile in the ratio 64:30:6 was selected as the best one and pumped at 1 mL/min at 40 °C. The use of this recent column and an aqueous mobile phase instead of buffers gives several advantages over the method herein developed; namely the fact that the chromatographic analysis takes only 9 min. The method was validated according to the guidelines of the Food and Drug Administration, showing to be accurate (bias within ±12%), precise (coefficient variation <9%), selective and linear (r ² > 0.997) over the concentration range of 0.05–30 μg/mL for carbamazepine; 0.05–20 μg/mL for oxcarbazepine; 0.15–4 μg/mL for eslicarbazepine acetate; 0.1–30 μg/mL for carbamazepine-10,11-epoxide; 0.1–10 μg/mL for 10,11-trans-dihydroxy-10,11-dihydro-carbamazepine, and 0.1–60 μg/mL for licarbazepine. It was also shown that this method can adequately be used for the therapeutic drug monitoring of the considered antiepileptic drugs, carbamazepine, oxcarbazepine, eslicarazepine acetate, and their metabolites.     

Development and validation of an agar diffusion assay for determination of ceftazidime in pharmaceutical preparations

Ceftazidime (CFZ) is a broad spectrum parenteral beta-lactam antibiotic of the cephalosporin family. This paper reports the development and validation of an agar diffusion microbiological assay using the cylinder-plate method for determination of CFZ in powder for injection. The validation carried out yielded good results in terms of linearity, precision, accuracy, selectivity, and robustness. The assay is based on the inhibitory effect of CFZ upon the strain of Pseudomonas aeruginosa ATCC 27853 used as the test microorganism. The results of the assays were treated statistically by analysis of variance and were found to be linear (correlation coefficient = 0.999998) in the selected range of 8.0-32.0 microg/mL; precise [repeatability: relative standard deviation (RSD) = 1.11%; intermediate precision: between-day RSD = 1.37% and between-analyst RSD = 1.41%]; and accurate. The selectivity of the bioassay was evaluated by analysis of degraded samples at 50 degrees C, and the results were compared with a pharmacopeial liquid chromatographic method at the time 0, 24, and 48 h. The results demonstrated the validity of the proposed bioassay, which allows reliable quantitation of CFZ in pharmaceutical samples and can be used as a useful alternative methodology for CFZ analysis in routine quality control.     

Development and validation of an immunochromatographic assay for rapid multi-residues detection of cephems in milk

A one-step immunochromatographic assay (ICA) was developed for the detection of seven kinds of cephems in milk. Polyclonal antibodies (PcAb) with group-specific to cephems were raised in rabbits after immunization with cephalexin-keyhole limpet hemocyanin (KLH) conjugate. The specificity of anti-sera was determined by indirect competitive enzyme-linked immunosorbent assay (icELISA), and the 50% inhibitions (IC₅₀) of cephalexin and cefadroxil were obtained at 1.5 ng mL⁻¹; IC₅₀ of cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were 4, 3.7, 3.2, 4.5 and 5 ng mL⁻¹, respectively. The PcAb against cephems were conjugated to colloidal gold particles as the detection reagent for ICA strips to test for cephems. This method achieved semi-quantitative detection of cephems in <5 min, with high sensitivity to cephalexin and cefadroxil (both 0.5 ng mL⁻¹). At the same time, cefatiofur, cefapirin, cefazolin, cefalothin and cefotaxine were detected at <100 ng mL⁻¹ in spiked processed-milk samples. This method was compared with an enzyme-linked immunosorbent assay by testing 40 milk samples, and the positive samples were validated by a high-performance liquid chromatographic method, with an agreement rate of 100% for both comparisons. In conclusion, the method was rapid and accurate for the multi-residue detection of cephems in milk.     

Development and validation of an assay method for the determination of trifluoroacetic acid in a cyclosporin-like drug

An improved method for the assay of trifluoroacetic acid (TFA) in a cyclosporin-like drug substance is presented, based on ion chromatography with suppressed conductivity detection. Column fouling by the drug molecule is avoided by use of a sample preparation method in which the drug substance is precipitated at alkaline pH whilst the TFA remains in solution. The new method requires a smaller sample mass than a previous method based on headspace-GC-FID whilst achieving an improvement in sensitivity. During validation, the method's performance was found to be consistent with usual acceptance criteria, and the method was found to be robust in routine use.     

The development and validation of a high performance liquid chromatography (HPLC)/tandem mass spectrometry assay for fenticonazole in human plasma and comparison with an HPLC-UV method

A method is described for the determination of fenticonazole in human female plasma. The method utilizes high performance liquid chromatography coupled to atmospheric pressure positive-ion chemical ionization triple quadrupole mass spectrometry. Multiple reaction monitoring is employed for selectivity and sensitivity which enables quantification over the range 0.5–20 ng mL⁻¹ with acceptable precision and accuracy. A comparison is made with an existing HPLC-UV assay and the utility of the technology of combined liquid chromatography and tandem mass spectrometry for subnanogram per mL assays is discussed.     

Development and validation of an HPLC/MS/MS method for the determination of sufentanil and morphine in human plasma

A sensitive and fast HPLC/MS/MS method for measurement of sufentanil and morphine in plasma was developed and validated. A single liquid-liquid extraction in alkaline medium was used for the cleanup of plasma, and fentanyl was added as an internal standard (IS). The analyses were carried out using a C18 column and the mobile phase acetonitrile-5 mM ammonium acetate + 0.25% formic acid (70 + 30, v/v). The triple-quadrupole mass spectrometer equipped with an electrospray source in positive mode was set up in the selective reaction monitoring mode to detect precursor --> product ion transition 387.0 > 238.0, 285.7 > 165.1, and 337.0 > 188.0 for sufentanil, morphine, and IS, respectively. The method was linear in the 0.05 (LOQ) - 500 ng/mL range for sufentanil and 10 (LOQ) - 1000 ng/mL range for morphine. Good selectivity, linearity, precision, accuracy, and robustness were obtained for the HPLC/MS/MS method. The proposed method was successfully applied for the determination of sufentanil and morphine in patients undergoing cardiac surgery.     

Development and validation of an LC-MS-MS method for determination of methyl kulonate in rat plasma

A sensitive, rapid and specific LC‐MS‐MS method was established and validated for determination of methyl kulonate, a major bioactive constituent isolated from Meliae Cortex, in rat plasma. Plasma samples were treated by precipitating protein with methanol and were chromatographed using a Capcell Pak C₁₈ column (100 × 4.6 mm, 5 µm) with the mobile phase comprising a mixture of methanol, 10 m m ammonium formate and formic acid (95:5:0.1, v/v/v). Detection and quantification were performed by mass spectrometry in the multiple reaction monitoring mode with positive atmospheric ionization at m/z 467 → 311 for methyl kulonate, and m/z 469 → 451 for dubione B (internal standard), respectively. A good linear response was observed over the concentration range 1.00–500 ng/mL with the lower limit of quantification 1.00 ng/mL in rat plasma. The method also afforded satisfactory results base on sensitivity, specificity, precision, accuracy, recovery, freeze–thaw and long‐time stability. The validated method was successfully applied to determine the pharmacokinetic properties of methyl kulonate in rats after oral administration at dose of 100 mg/kg. This pharmacokinetic study of methyl kulonate is reported here for the first time. Copyright © 2011 John Wiley & Sons, Ltd.     

Development,validation and analytical error function of two chromatographic methods with fluorimetric detection for the determination of bisoprolol and metoprolol in human plasma

This work describes two high-performance liquid chromatographic methods for the individual determination of bisoprolol and metoprolol in human plasma. Analytical methods involve two different liquid-liquid extractions of human plasma, with diethyl ether for bisoprolol and with dichloromethane for metoprolol, coupled with a similar Nucleosil C(18) reversed-phase HPLC column. Fluorimetric detection was used to identify both beta-blockers. Retention times for bisoprolol and metoprolol were 8.7 and 3.2 min, respectively. Linear regressions for the calibration curves were linear at a concentration range of 6.25-200 ng/mL. Intra- and inter-day precision coefficients of variations and accuracy bias were acceptable (within 15%) over the entire range for both drugs. Average recovery was 89% for metoprolol and 98% for bisoprolol. Once the methods had been validated, analytical error functions were established as standard deviation (SD; ng/mL) = 2.216 + 3.608 x 10(-4)C(2) (C = theoretical concentration value) and SD-(ng/mL) = 0.408 + 0.378 x 10(-1)C for bisoprolol and metoprolol, respectively. The methods developed and their associated analytical error functions will be suitable for pharmacokinetic studies and for determination of plasma concentration if posology individualization of these drugs is needed.     

Automated capillary gas chromatographic assay using nitrogen-phosphorus ionization detection for the determination of bepridil in human plasma

A highly sensitive and specific capillary gas chromatographic method for the determination of the antianginal drug bepridil in plasma is described. The capillary gas chromatograph and nitrogen-selective detector combination provides excellent sensitivity for clinical samples. The lowest concentration of bepridil which can be measured accurately and precisely in a 1-ml plasma sample is 1 ng/ml. Standard curves are linear over the concentration range 1-60 ng/ml. Accuracy and precision of the assay, expressed as relative deviation from the true value and relative standard deviation (inter-run) are less than 15% at all concentrations in the linear range. No interfering peaks are observed. Using an automatic injector and a laboratory computer system, sixty samples can be analyzed routinely in one day. The present assay has been successfully cross-validated with a published high-performance liquid chromatographic assay.     

Development and validation of a liquid chromatographic method for the determination of hydroxymethylfurfural and alpha-ketoglutaric acid in human plasma

Hydroxymethylfurfural (HMF) and alpha-ketoglutaric acid (KG) have been recently investigated as potential cancer cell damaging agents. We herein report for the first time a validated quantitative assay for their simultaneous determination in human plasma which is amenable to be applied in the future screening of the target compounds in human probands in order to properly design a targeted chemotherapeutic regimen for certain types of malignant tumors.A simple liquid chromatographic method in conjunction to derivatization after a two-step optimized solid phase clean-up procedure is described. The method is based on the reaction of HMF and KG with 2-nitrophenylhydrazine or 2,4-dinitrophenylhydrazine in an aqueous environment. Reaction conditions were studied with respect to pH, reagent volume, reaction temperature and time. Exact testing of such parameters beside careful selection of the mobile phase composition rendered feasible the quantification of the chemically significantly differing analytes along a single chromatographic run. The formed derivatives could be separated isocratically by reversed-phase LC on a C₈-column. Detection in the UV and in the visible range is possible. Results showed good recovery and reproducibility with detection limits (S/N = 3) down to 2 picomoles analyte on column. Resolution of the syn and anti geometric isomers of the HMF and KG derivatives is possible. The isomeric ratio in relation to the reaction pH is discussed.     

Development and validation of a LC-MS/MS method for d-cycloserine determination in human plasma for bioequivalence study

A reliable and high throughput high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for determining levels of the antitubercular drug-d -cycloserine in human plasma. Plasma samples analyte with an internal standard (IS) (niacin) were prepared by solid-phase extraction using Waters Oasis MCX cartridges. The chromatographic separation was performed using the HILIC mode on a YMC-Pack SIL-06 column (150?×?4.6 mm; 3 μm) under isocratic conditions. The run time of analysis was 5 min. The mobile phase consisted of methanol, propanol-2 and 0.075 % trifluoroacetic acid (66.5:28.5:5, v/v/v). Protonated ions formed by turbo ion spray in positive mode were used to detect the analyte and the IS. MS/MS detection was used to monitor the fragmentation of 103–75?m/z for cycloserine and 124 to 80?m/z for niacin (IS) on an API 4000 (AB Sciex) triple quadrupole mass spectrometer. A linear dynamic range of 0.3–30 μg/mL was established for cycloserine using 0.2 mL human plasma and a 1 μL injection volume. The mean relative recovery of cycloserine and niacin were 77.2 and 82.4 %, respectively. The procedure of sample preparation was consistent and reproducible (precision, 0.8–3.4 %; accuracy, 93.8–104.9 %). The method was validated in accordance with requirements of the European Medicines Agency and successfully applied to a bioequivalence study of 250 mg tablet formulations in 23 healthy human subjects.     

Development of an HPLC method for the determination of anidulafungin in human plasma and saline

An ultraviolet high-performance liquid chromatography (HPLC) method was developed to analyze anidulafungin in human plasma and saline. A reversed-phase column was used with a UV detector set at 310 nm. The mobile phase consisted of methanol and ammonium phosphate buffer at a flow rate of 1 mL/min. Micafungin was used as the internal standard. Both standard curves were linear over a range of 1 to 10 μg/mL. The intra-assay relative standard deviations (RSD) for plasma and saline matrices were 1.60-1.81% and 1.96-3.70%, respectively. The inter-assay RSD for plasma and saline matrices were 2.41-7.25% and 1.31-3.16%, respectively. This method successfully recapitulated anidulafungin plasma concentrations previously analyzed by HPLC-tandem mass spectrometry with precision and accuracy of 6.9% and 1.59%, respectively.     

Development and validation of liquid chromatography–mass spectrometry method for the determination of telmisartan in human plasma

A sensitive liquid chromatographic–electrospray ionization mass spectrometric method was developed and validated for fast determination of telmisartan in human plasma. Plasma of 0.1 mL was deprotienated with methanol, centrifugation, evaporation to dryness and dissolving in mobile phase, samples were separated using a Hypersil-Keystone C18 reversed-phase column (150 mm × 2.1 mm i.d., 5 μm), together with a mobile phase containing of acetonitrile–10 mM ammonium acetate (42:58, v/v), 0.2% acetic acid and was isocratically eluted at a flow rate of 0.2 mL/min. Telmisartan and its internal standard, valsartan, were measured by electrospray ion source in positive selective ion monitoring mode. The method demonstrated linearity from 1 to 2000 ng/mL (r = 0.9988). The limit of quantification for telmisartan in plasma was 1 ng/mL with good accuracy and precision. The mean sample extract recovery of the method were higher than 82 and 78% for telmisartan and internal standard (IS), respectively. The within-run and between-run precision ranged from 3.4 to 8.9% and 5.9 to 11.2% (relative standard deviation, R.S.D.), respectively.     

Development and validation of a simple and efficient HPLC method for the determination of zonisamide in pharmaceuticals and human plasma

A simple and efficient liquid chromatographic method has been developed and validated for the determination of zonisamide in pharmaceuticals and human plasma. Plasma samples are analyzed after one step protein precipitation with methanol, and chromatographic separation of zonisamide and chloramphenicol (internal standard) is carried out using a C₁₈ column and the optimum mobile phase of acetonitrile/methanol/distilled water (20: 10: 70, v/v/v). The method is validated in both mobile phase and human plasma, and the obtained limits of quantification values are 0.099 and 0.12 μg/mL in mobile phase and human plasma, respectively. Fully validated method is reproducible and selective for the determination of zonisamide in pharmaceuticals and human plasma.     

HPLC-UV method development and validation for the determination of low level formaldehyde in a drug substance

A reversed-phase high-performance liquid chromatographic method (HPLC) with diode-array detection is developed and validated for the quantitative determination of formaldehyde in a drug substance. Formaldehyde (HCHO) is reacted with 2,4-dinitrophenylhydrazine (DNPH) to form a Schiff base (HCHO-DNPH derivatization product), which has an absorbing maximum (lambda max) at 360 nm. The HPLC method employs a C8, 3-microm particle size analytical column (150 mm x 4.6 mm), 15-microL injection volume, column temperature controlled at 30 degrees C, detection at 360 nm, and a water-acetonitrile (55:45, v/v) mobile phase at a flow rate of 1 mL/min. These conditions resolve the HCHO-DNPH product from unreacted DNPH, the drug substance and related impurities, as well as diluent peaks within 20 min. The retention time of the HCHO-DNPH product is approximately 6.4 min. The method is linear, accurate in the specified range (0.33-333 ppm), and robust based on analyte (HCHO-DNPH derivatization product) stability in standard and sample. Detection limit is 0.03 ng (0.1 ppm).     

Development and validation of an LC‐APCI‐MS/MS method for the determination of phenethyl isothiocyanate in human plasma

Phenethyl isothiocyanate (PEITC) is a promising chemopreventive agent present in cruciferous vegetables. This paper describes the development of a method for the determination of PEITC in human plasma by liquid chromatography/tandem mass spectrometry (LC‐MS/MS). Atmospheric‐pressure chemical ionization was found more suitable for ionization of PEITC than electrospray ionization. Because of the lability of PEITC, a combination of low temperature and acidification was applied to minimize the degradation during the sample collection and preparation procedure. A simple protein precipitation with acetonitrile was used for the preparation of plasma samples. The analyte and 1‐phenylpropyl isothiocyanate as internal standard (IS) were subjected to chromatographic analysis on a C₁₈ column (50 × 2.1 mm, 5 µm) using 85% methanol as mobile phase at a flow rate of 0.3 mL/min. The total analysis time for each chromatograph was 3 min and the results were linear over the studied range (5.00–250 ng/mL). The intra‐ and inter‐day precision values were acceptable as per US Food and Drug Administration guidelines. This method was successfully applied in the determination of PEITC concentrations in plasma samples from healthy chinese Volunteers. Copyright © 2014 John Wiley & Sons, Ltd.