Biomedical Chromatography

Srinivas, Nuggehally R.

doi: 10.1002/bmc.447pmid: 15584016

The field of chiral separations had a modest beginning some two decades ago. However, due to rapid technological advancement coupled with simultaneous availability of innovative chiral stationary phases and novel chiral derivatization agents, the field of chiral separations has now totally outpaced many other separation fields. Keeping pace with rapid changes in the field of chiral separations, investigators continue to add stereoselective pharmacokinetic, pharmacodynamic, pharmacologic and toxicological data of new and/or marketed racemic compounds to the literature. Examination of the evolution of chiral separations suggests that in the beginning many investigators attempted to separate and quantify a single pair of enantiomers, adopting either direct (separation made on a chiral stationary phase) or indirect (separation made following precolumn conversion of enantiomers to corresponding diastereomers) approaches. However, more recent trends in chiral separations suggest that investigators are attempting to separate and quantify multiple pairs of enantiomers with available technologies. Added to this, some interesting trends have been observed in many of the recently reported chiral applications, including preferences regarding internal standard selection, mobile phase contents and composition, sorting out issues with mass spectrometric detection, determination of elution order, analytical manipulations of metabolite(s) without reference standards and addressing some specificity‐related issues. This review mainly focuses on chiral separations involving multiple chiral analytes and attempts to justify the need for such chiral separations involving multiple analytes. In this context, several cases studies are described on the utility and applicability of such chiral separations under discrete headings to provide an account to the readership on the implications of such tasks. The topics of case studies covered in this review include: (a) therapy markers—differentiation from drug abuse and/or applicability in forensics; (b) role in pharmacogenetic/polymorphic evaluation; (c) monitoring and understaning the role of parent and active metabolite(s) in clinical and preclinical investigations; (d) exploration on the pharmacokinetic utility of an active chiral metabolite vis‐à‐vis the racemic parent moiety; (e) understanding the chirality play in delineating peculiar toxic effects; (f) exploration of chiral inversion phenomenon, and understanding the role of stereoselective metabolism. For the further benefit of readership, some select examples (n = 19) of the separation of multiple chiral analytes with appropriate information on chromatography, detection system, validation parameters and applicable conclusion are also provided. Finally, the review covers some useful considerations for method development involving multiple chiral analytes. Copyright © 2004 John Wiley & Sons, Ltd.

Sheng, Yuxin; Li, Lie; Zhang, Jinlan; Guo, Dean

doi: 10.1002/bmc.389pmid: 15386578

A high performance liquid chromatographic method (HPLC), together with solid phase extraction (SPE), was developed for simultaneous determination of albiflorin and paeoniflorin in rat urine after oral administration of Si–Wu decoction. The samples were pretreated with solid phase extraction using Extract‐Clean™ cartridges. Analysis of the extract was performed on a reversed‐phase C18 column and a mobile phase made up of acetonitrile and 0.03% formic acid (17:83, v/v). UV detection was set at 230 nm. The assay was linear over the range 2.625–52.50 mg/mL for albiflorin and 3.875–77.50 µg/mL for paeoniflorin. The average percentage recoveries of three spiked urines were 97.01 ± 3.32 and 102.32 ± 6.97 for albiflorin and paeoniflorin, respectively. The intra‐day precision (RSD) ranged from 0.21 to 1.79% at concentrations of 4.20, 10.50, 26.25 and 39.375 µg/mL of albiflorin and 0.12 to 2.92% at concentrations of 3.875, 10.85, 23.25 and 58.125 µg/mL of paeoniflorin, and inter‐day precision (RSD) was from 1.02 to 1.86% for albiflorin and 0.94 to 3.30% for paeoniflorin, at the same four concentrations. This method was applied in order to analyze albiflorin and paeoniflorin in rat urine following oral administration of traditional Chinese medicinal preparation of Si–Wu decoction. Copyright © 2004 John Wiley & Sons, Ltd.

Assimopoulou, A. N.; Papageorgiou, V. P.

doi: 10.1002/bmc.390pmid: 15386577

The chiral pair alkannin and shikonin (A/S) are potent pharmaceutical substances with a wide spectrum of biological activity; their enantiomeric ratio does not influence the major biological activity studied hitherto. Nevertheless, in pharmaceutical development and approval of chiral drugs from the Health and Regulatory Authorities, full documentation of methods of analysis of enantiomeric drugs, is required in order to evaluate the enantiomeric purity of starting materials and final products and to control the stability of enantiomers in pharmaceutical formulations under several experimental conditions. In the present study, the enantiomeric ratio of A/S was determined in several commercial samples of alkannin and shikonin and also the proportion of A/S derivatives in several Alkanna root samples, which are all used as active ingredients in pharmaceuticals. Light and air proved not to influence the enantiomeric ratio of A/S on a shikonin commercial sample, and temperature also did not alter the A/S ratio on shikonin and alkannin commercial samples. Microencapsulation of alkannin and shikonin commercial samples in ethylcellulose microspheres and also molecular inclusion of a shikonin commercial sample in β‐hydroxypropyl‐cyclodextrin, which are used as drug delivery systems, did not alter the A/S enantiomeric ratio. Copyright © 2004 John Wiley & Sons, Ltd.

Kanaze, Feras Imad; Bounartzi, Melpomeni I.; Niopas, Ioannis

doi: 10.1002/bmc.391pmid: 15386576

Diosmetin, 3′,5,7‐trihydroxy‐4′‐methoxyflavone, is the aglycone of the flavonoid glycoside diosmin that occurs naturally in foods of plant origin. Diosmin exhibits antioxidant and anti‐inflammatory activities, improves venous tone and it is used for the treatment of chronic venous insufficiency. Diosmin is hydrolyzed by enzymes of intestinal microflora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the determination of diosmetin in human plasma was developed and validated. Diosmetin and the internal standard 7‐ethoxycoumarin were isolated from plasma by liquid–liquid extraction and separated on a C8 reversed‐phase column with methanol–water–acetic acid (55:43:2, v/v/v) as the mobile phase at 43°C. Peaks were monitored at 344 nm. The method was linear in the 10–300 ng/mL concentration range (r > 0.999). Recovery for diosmetin and internal standard was greater than 89.7 and 86.8%, respectively. Intra‐day and inter‐day precision for diosmetin ranged from 1.6 to 4.6 and from 2.2 to 5.3%, respectively, and accuracy was better than 97.9%. Copyright © 2004 John Wiley & Sons, Ltd.

Beaudry, Francis; Proulx, Dave; Furtado, Milton

doi: 10.1002/bmc.393pmid: 15386575

A fast and sensitive method has been developed and validated for the determination of bucillamine in human blood by derivatizing the free sulfhydryl groups with isobutyl acrylate (IA), by APCI‐LC/MS/MS. The collected blood sample was immediately mixed with a mixture of IA and 0.05 m Tris(hydroxymethyl)aminomethane hydrochloride (Tris–HCl) buffer, pH 9.2, to stabilize the sulfyhydryl moieties. The derivatized samples were then extracted by protein precipitation, evaporated, reconstituted and injected using an LC‐APCI/MS/MS instrument. Separation was achieved using a C18 analytical column and a gradient mobile phase within a chromatographic run time of 5 min. A quadratic (weighted 1/concentration2) relationship was observed during validation over a concentration range of 0.4–40 µg/mL with a correlation value of r ≥ 0.9966. The inter‐batch precision and accuracy at low, medium and high concentrations were 8.1, 8.4 and 7.3%; 113.3, 104.9 and 103.9%, respectively, and the intra‐batch precision and accuracy at low, medium and high concentrations were 7.7, 5.4 and 2.7%; 105.1, 111.9 and 113.2%, respectively. Copyright © 2004 John Wiley & Sons, Ltd.

Fukushima, Takeshi; Kawai, Junko; Imai, Kazuhiro; Toyo'oka, Toshimasa

doi: 10.1002/bmc.394pmid: 15386574

Both d‐ and l‐serine in rat brain microdialysis sample were simultaneously determined by pre‐column fluorescence derivatization with 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), separation of the derivatives on ODS column, TSKgel ODS‐80TsQA, followed by Pirkle type chiral columns, Sumichiral OA‐2500 (S), which gave a sufficient enantiomeric separation of NBD–d‐serine and NBD‐l‐serine, and fluorimetric detection at a wavelength of 540 nm with an excitation wavelength of 470 nm. The peaks of NBD–d‐serine and NBD–l‐serine in the rat brain microdialysis sample were clearly found, and the validation study showed satisfactory results; the precision and accuracy were within 5.14 and 109%, respectively. Using the proposed HPLC method, the time‐course profile of d‐serine concentration in rat prefrontal cortex following intraperitoneal administration of d‐serine was investigated. As a consequence, d‐serine appeared to be rapidly distributed in the brain, and then decreased gradually with time in the extracellular fluid of the rat prefrontal cortex. The proposed HPLC method will be useful for in vivo studies on d‐serine, which acts as a coagonist for N‐methyl‐d‐aspartate receptor, to the extracellular fluid of rat brain. Copyright © 2004 John Wiley & Sons, Ltd.

Wang, Ching‐Chiung; Chen, Fu‐An; Chen, Chih‐Jui; Chao, Su‐Hui; Wu, An‐Bang

doi: 10.1002/bmc.395pmid: 15386573

A sample of 10 mm zomepirac in methanol was photo‐irradiated with a Hanovia 200 W high‐pressure quartz Hg lamp for 14 days. In total, four photoproducts were observed from the HPLC chromatogram. The preparative HPLC included an YMC‐Pack Pro C18 column (250 × 20 mm i.d.), a mobile phase of CH3CN–CH3OH–1%HOAc (10:60:30, v/v/v), and UV detection at 254 nm. The most probable structures of the four photoproducts were determined by LC‐MS. Two major photoproducts were separated, and their structures were further confirmed by the spectroscopic methods. A reaction scheme of zomepirac was proposed that the photochemical reaction routes occur mainly via bond fission between carbonyl–pyrrolyl groups (α‐cleavage of a ketone), and decarboxylation followed by oxidation with singlet oxygen to produce an aldehyde. Copyright © 2004 John Wiley & Sons, Ltd.

Gao, Wenhua; yaowen, Chen; Yin, Yegao; Chen, Xingguo; Hu, Zhide

doi: 10.1002/bmc.396pmid: 15452858

A novel microemulsion electrokinetic chromatography (MEEKC) method for separating and determining two sesquoterpene lactones, alantolactone (AL) and isoalantolactone (IAL), in Radix inulae and Liuwei Anxian San has been developed. The effects of several important factors such as internal organic phases, concentration of microemusion, concentration of acetonitrile, injection time and running voltage were systematically investigated to determine the optimum conditions. The optimum microemulsion system was composed of n‐hexane (0.32% w/w), SDS (1.24% w/w), 1‐butanol (2.64% w/w), acetonitrile (10% w/w) and 10 mm sodium tetraborate buffer (85.80% w/w, pH 9.2). The applied voltage was 20 kV. The analytes were detected at 214 nm. Regression equations revealed linear relationships (correlation coefficients 0.9950 for AL and 0.9946 for IAL) between the peak area of each analyte and the concentration. The limits of detection (defined as a signal‐to‐noise ratio of about 3) were approximately 0.45 µg/mL for AL and 0.56 µg/mL for IAL. The levels of the analytes were successfully determined with recoveries ranging from 98.2 to 104.3%. Furthermore, a simple and effective extraction method, with methanol in an ultrasonic water bath for 60 min, was used for sample preparing. Also, MEEKC was compared with micellar electrokinetic chromatography (MEKC) and shown better separation results. Copyright © 2004 John Wiley & Sons, Ltd.

Yu, Lushan; Luan, Lianjun; Shao, Qing; Zeng, Su

doi: 10.1002/bmc.397pmid: 15386572

Propranolol, available commercially as a racemic mixture, is a non‐selective β‐adrenergic blocking agent used in the treatment of hypertension, angina pectoris and cardic arrhythmias. We have developed and validated an RP‐HPLC assay method for direct determination of R‐(+)‐ and S‐(−)‐propranolol glucuronide in rat hepatic microsomes to investigate the enantioselectivity of propranolol glucuronidation metabolism. A baseline separation of propranolol glucuronide enantiomers was achieved on a 5 µm reversed‐phase ODS column, with a mixture of phosphate buffer (pH 3.5, 0.067 mol/L) and methanol (55:45, v/v) as mobile phase. Ultraviolet detection was set at 220 nm, and p‐nitrobenzoic acid was used as internal standard. The standard curve of assay for R‐(+)‐ and S‐(−)‐propranolol glucuronide in spiked microsomal incubate showed good linearity throughout the concentration range from 0.50 to 20.0 µmol/L. The analytical method affords average recovery of 99.8 and 100.1% for R‐(+)‐ and S‐(−)‐propranolol glucuronide, respectively. The method provides a high sensitivity and good precision for R‐(+)‐ and S‐(−)‐propranolol glucuronide (RSD < 10%). The LOD was 0.15 µmol/L and the LOQ was 0.5 µmol/L (RSD < 8%, n = 5) for both R‐(+)‐ and S‐(−)‐propranolol glucuronide. The method is simple, precise and accurate, and is suitable for quantifying the propranolol glucuronides enantiomers in rat hepatic microsomes. Copyright © 2004 John Wiley & Sons, Ltd.

Bhushan, R.; Gupta, Deepak

doi: 10.1002/bmc.398pmid: 15386571

The enantiomeric resolution of (±)‐ibuprofen into its enantiomers was achieved by TLC on silica gel plate using optically pure (−)‐brucine as a chiral selector and acetonitrile–methanol (5:1, v/v) as the solvent system. Spots were located in an iodine chamber. The detection limit was 4.9 µg. The effect of concentration of the chiral selector, temperature and pH on resolution has been studied. Copyright © 2004 John Wiley & Sons, Ltd.