Biomedical Chromatography

Wattananat, Triporn; Akarawut, Wiyada

doi: 10.1002/bmc.538pmid: 15999337

A high‐performance liquid chromatographic assay with UV detection was developed for the determination of fluconazole in human plasma. The method utilized solid‐phase extraction for sample clean‐up. The separation was performed on a C18 column by isocratic elution with a mobile phase of 10 mM acetate buffer at pH 5.0 and methanol and UV detection at 210 nm. Validation was performed according to the current recommendations of the USFDA bioanalytical method validation guidance. The method proved to be specific, accurate, precise and linear between 200 and 10,000 ng/mL with correlation coefficients greater than 0.999. The coefficient of variation was within 11% and relative deviation was less than 10%. Copyright © 2005 John Wiley & Sons, Ltd.

Kowalska, Sylwia; Krupczyńska, Katarzyna; Buszewski, Bogusław

doi: 10.1002/bmc.543pmid: 16041787

Biologically active compounds such as vitamins, steroids, nucleosides, peptides and proteins play a very important role in coordinating living organism functions. Determination of those substances is indispensable in pathogenesis. Their complex structure and physico‐chemical properties cause many analytical problems. Chromatography is the most common technique used in pharmaceutical and biomedical analysis. The interaction between analyte and stationary phase plays a major role in the separation process. The structure of the packing has a significant influence on the results of the separation process. Various types of spectroscopic techniques, such as nuclear magnetic resonance spectroscopy, infrared spectroscopy, fluorescence spectroscopy and photoacoustic spectroscopy can be useful tools for the characterization of packings. Surface area measurements, elemental analysis, thermal analysis and microcalorimetric measurements are also helpful in this field. Part of the paper contains a description of chromatographic tests used for the determination of column properties. The description of the possibilities of surface characterization is not complete, but is focused on the most popular techniques and practical chromatographic tests. All the presented methods made possible the design and quality control of a new generation stationary phases, which are the future of high‐performance liquid chromatography. Copyright © 2005 John Wiley & Sons, Ltd.

Silverthorn, Courtney F.; Parsons, Teresa L.

doi: 10.1002/bmc.519pmid: 15920701

A fully validated and clinically relevant assay was developed for the assessment of nevirapine concentrations in neonate blood plasma samples. Solid‐phase extraction with an acid–base wash series was used to prepare subject samples for analysis. Samples were separated by high performance liquid chromatography and detected at 280 nm on a C8 reverse‐phase column in an isocratic mobile phase. The retention times of nevirapine and its internal standard were 5.0 and 6.9 min, respectively. The method was validated by assessment of accuracy and precision (statistical values <15%), specificity, and stability. The assay was linear in the range 25–10,000 ng(sol )mL (r2 > 0.996) and the average recovery was 93% (n = 18). The lower limit of quantification (relative standard deviation <20%) was determined to be 25 ng(sol )mL for 50 µL of plasma, allowing detection of as little as 1.25 ng of nevirapine in a sample. This value represents an increase in sensitivity of up to 30‐fold over previously published methods. Copyright © 2005 John Wiley & Sons, Ltd.

Hirabayashi, Yoshihiro; Tsuchiya, Kiyoto; Kimura, Satoshi; Oka, Shinichi

doi: 10.1002/bmc.521pmid: 15971289

We report the development of a simple, economical and reliable chromatographic method for the simultaneous determination of six HIV protease inhibitors (PIs; amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir), the active metabolite of nelfinavir (M8) and the non‐nucleoside reverse transcriptase inhibitor (NNRTI; efavirenz) in human plasma. After extraction from plasma with an ethyl acetate–acetonitrile mixture, the analytes were separated on a phenyl column with a gradient of acetonitrile and phosphate solutions, and detected at three ultraviolet wavelengths. Calibration curves were linear over the range 0.025–15 µg(sol )mL for saquinavir and 0.05–15 µg(sol )mL for the other analytes. The accuracies ranged from −6.9% to +7.6%, and the intra‐assay and inter‐assay precisions were <9.2 and <11.8%, respectively. Our method, covering most of the PIs and NNRTIs currently used, facilitates ready therapeutic drug monitoring in hospital laboratories. Copyright © 2005 John Wiley & Sons, Ltd.

Shen, Xiaoxuan; Boswell, C. Andrew; Wong, Edward H.; Weisman, Gary R.; Anderson, Carolyn J.; Tomellini, Sterling A.

doi: 10.1002/bmc.525pmid: 15954159

The use of copper radioisotopes in imaging and therapy has prompted an increased interest in chelators which form stable copper complexes, such as Cu(II)–azamacrocyclic complexes. The effects of charge, stability and the size of the macrocyclic backbone of the Cu(II)–azamacrocyclic complexes on biological behavior have been evaluated. Here we report a reversed‐phase high‐performance liquid chromatography (HPLC) method to separate several Cu(II)–azamacrocyclic complexes, including Cu(II) complexes of 1,4,8,11‐tetraazacyclotetradecane‐1,4,8,11‐tetraacetic acid (TETA), 4,11‐bis(carboxymethyl)‐1,4,8,11‐tetraazabicyclo(6.6.2)hexadecane (CB‐TE2A) and 4,10‐bis(carboxymethyl)‐1,4,7,10‐tetraazabicyclo(5.5.2)tetradecane (CB‐DO2A). Absorbance at 280 nm was used to monitor the complexes as they eluted from the reversed‐phase column. The effects of the concentration of the buffer, the pH of the buffered mobile phase and the concentration of the organic modifier, methanol, on the separation were investigated. Separation of these copper complexes by ion‐pair HPLC with the use of a mass spectrometry‐compatible ion‐pair reagent, triethylammonium acetate, in the mobile phase at pH 6.3 is also presented. The reversed‐phase chromatographic conditions utilized also allow the pKas of Cu–TETA and the log(k′w) values of Cu–CB‐TE2A, Cu–TETA and Cu‐CB‐DO2A to be estimated. Copyright © 2005 John Wiley & Sons, Ltd.

Zhou, Xiao‐fei; Wang, Qi; Coburn, Robert A.; Morris, Marilyn E.

doi: 10.1002/bmc.526pmid: 15954160

A simple and reliable reversed‐phase high‐performance liquid chromatography method was developed and validated for the determination of DHP‐014, a niguldipine analogue with potent P‐glycoprotein inhibitory and negligible calcium channel blocking properties, in rat plasma. DHP‐014 and niguldipine hydrochloride (the internal standard) were extracted from rat plasma by liquid extraction using hexane. DHP‐014 was then separated by HPLC on a C18 column and quantified by ultraviolet detection at 238 nm. The mobile phase consisted of acetonitrile–aqueous 5 mm phosphate buffer (65:35, v(sol )v) containing 0.4% (v(sol )v) triethylamine adjusted to pH 7.0. The mean extraction efficiency of DHP‐014 was 109.0 ± 12.9, 97.7 ± 8.0 and 102.9 ± 7.5% for DHP‐014 concentrations of 10, 50 and 100 nm, respectively (n = 5). The method was linear over the concentration range 2.5–200 nm with a regression coefficient of 0.998. The limit of detection of DHP‐014 in rat plasma was 1.0 nm. The intra‐ and inter‐day coefficients of variation for DHP‐014 in rat plasma were 4.7–7.9 and 6.9–9.9%, respectively. The intra‐ and inter‐day accuracy was 98.2–99.5 and 97.9–103%, respectively. The bioanalytical technique was used to determine DHP‐014 in plasma samples in a pharmacokinetic study of DHP‐014 administered to female Sprague–Dawley rats. Copyright © 2005 John Wiley & Sons, Ltd.

Al‐Dirbashi, Osama Y.; Jacob, Minnie; Al‐Hassnan, Zuhair; Chabayta, Reem W.; El‐Badaoui, Fahad; Rashed, Mohamed S.

doi: 10.1002/bmc.527pmid: 15966058

We developed and validated an HPLC method with intramolecular excimer‐forming fluorescence derivatization to determine methylmalonic acid, a unique biochemical marker for methylmalonic aciduria. Methylmalonic acid in urine and an internal standard were derivatized with pyrenebutyric hydrazide and separated on a C8 column. The derivatives were detected by monitoring the fluorescence at 475 nm (excitation wavelength 345 nm). At a signal‐to‐noise ratio of 3, the detection limit was 0.33 pmol on the column and the calibration curve was linear up to 1 mmol(sol )L in urine. In a retrospective study on a relatively large number of known methylmalonic aciduria cases (n = 48), the method enabled us to differentiate methylmalonic aciduria cases from healthy controls (n = 52), regardless of age of patients at sampling or years of specimen storage. No interference was observed from isomeric or other dicarboxylic acids, or other urine constituents. As described, the method can be used retrospectively or prospectively for the diagnosis of methylmalonic aciduria and can be easily adopted by laboratories with no access to gas chromatography–mass spectrometry. Copyright © 2005 John Wiley & Sons, Ltd.

Guo, Ji‐fen; Zhang, Ai‐jun; Zhao, Ling; Sun, Xiao‐hong; Zhao, Yi‐min; Gao, Hong‐zhi; Liu, Ze‐yuan; Qiao, Shan‐yi

doi: 10.1002/bmc.528pmid: 15954161

A sensitive liquid chromatographic–tandem mass spectrometry(LC–MS(sol )MS) method was developed for the determination of rizatriptan in human plasma. The analytes were extracted from plasma samples by liquid–liquid extraction, separated on a Zorbax XDB C8 column (150 × 4.6 mm i.d.) and detected by tandem mass spectrometry with an electrospray ionization interface. Zomitriptan was used as the internal standard. The method had a lower limit of quantitation of 50 pg(sol )mL for rizatriptan, which showed more sensitivity and speed of analysis compared with reported methods. The within‐ and between‐day precision was measured to be below 11.71% and accuracy between −5.87 and 0.86% for all quality control samples. This quantitation method was successfully applied to the evaluation of the pharmacokinetic profiles of rizatriptan after single oral administration of 5, 10 and 15 mg rizatriptan tablets to 10 healthy volunteers (five males and five females). Copyright © 2005 John Wiley & Sons, Ltd.

Wei, Gang; Ding, Ping‐Tian; Zheng, Jun‐Min; Lu, Wei‐Yue

doi: 10.1002/bmc.529pmid: 15954162

In order to develop a thermosetting gel‐based formulation, the ocular pharmacokinetics of timolol was studied utilizing microdialysis sampling technique after topical administration. A linear microdialysis probe was characterized and implanted in the anterior chamber of a rabbit. Dialysate samples collected from the aqueous humor (AH) were directly injected into the HPLC system without any pre‐treatment and no interference was observed in the blank sample. The measured in vitro recovery of the probe was 57.67%; however, the in vivo recovery significantly decreased to 16.78% when assessed by the retrodialysis method, which was used to calculate the timolol concentration in AH. Although in the initial 15 min the drug concentrations in AH were comparable to that of the timolol solution, increased Cmax and significantly improved ocular bioavailability were obtained for the gel. When sodium deoxycholate (DC) was incorporated in the gel as a penetration enhancer, a 2‐fold increment in the ocular bioavailability was achieved with an increased Cmax and significantly suspended Tmax. The results demonstrated that microdialysis coupled to HPLC is a powerful tool to investigate the ocular pharmacokinetic, and hence facilitates the design of ophthalmic formulations. Copyright © 2005 John Wiley & Sons, Ltd.

Sparidans, Rolf W.; Dost, Frits; Crommentuyn, Kristel M. L.; Huitema, Alwin D. R.; Schellens, Jan H. M.; Beijnen, Jos H.

doi: 10.1002/bmc.530pmid: 15954163

Atazanavir is the most recently introduced protease inhibitor for the suppression of the anti‐human immunodefficiency virus. A sensitive and selective reversed‐phase liquid chromatographic assay for this drug in human plasma has been developed and validated. Atazanavir was isolated from a 500 µL plasma sample using liquid–liquid extraction with dichloromethane. After evaporation and reconstitution of the extract the sample was analysed using liquid chromatography and ultraviolet detection at 280 nm. In the evaluated concentration range (44–4395 ng(sol )mL atazanavir), intra‐day precisions were ≤7% and inter‐day precisions were ≤14%. Accuracies between 96 and 106% were found. The lower limit of quantification was 44 ng(sol )mL with an intra‐day precision of 7%, an inter‐day precision of 14% and an accuracy of 87%. There was no interference from 32 tested potentially co‐administrated drugs and metabolites. The usefulness of the assay was demonstrated for samples obtained from an HIV‐infected patient treated with atazanavir. Copyright © 2005 John Wiley & Sons, Ltd.