Reversed-phase ion-pair liquid chromatographic method for the determination of low concentrations of haloperidol in plasma

A sensitive liquid chromatographic method for the determination of haloperidol in plasma is described. The efficient and simple extraction procedure, followed by reversed-phase ion-pair liquid chromatography on a 3-micron octadecylsilica column and UV absorbance detection, makes it possible to determine concentrations down to 0.5 nmol/l with acceptable precision. In a pharmacokinetic study, in which 5 mg of haloperidol were given orally, the plasma levels were followed for 48 h.     

Reversed-phase liquid chromatographic method for the determination of ochratoxin A in wine

In this paper, we propose a new, rapid, highly sensitive and reproducible RP-HPLC-FLD method for the detection of ochratoxin A (OTA) in wine, by directly injecting the liquid in the chromatographic system without any extraction or clean-up. An alkaline mobile phase (NH4Cl:CH-CN 85:15 (v/v), 20 mM, pH 9.8) was used to obtain a distinct fluorescence enhancement. This improvement allows to reach, without an immunoaffinity clean-up or concentration, a detection limit of 0.05 ng/ml, which is similar to those commonly obtained after immunoaffinity purification and acidic elution. The method was statistically validated and directly applied to a series of wine samples.     

Reversed-phase liquid chromatographic method for estrogen determination in equine biological samples

Equine unsaturated estrogens are the main components of brand formulations indicated for hormonal replacement therapy in both hypogonadic and postmenopausal women. These hormones are produced by the fetoplacental unit during equine gestation. A method is described for the quantitative determination of equilenin (EL), equilin (EQ), 17alpha-dihydroequilin (17dEQ), and estrone (El) in the plasma of a pregnant mare. Blood samples are obtained weekly during pregnancy by jugular venipuncture using sodium ethylenediaminetetracetic as the anticoagulant. For the quantitation of these estrogens, plasma is submitted to enzymatic hydrolysis followed by liquid-liquid extraction. A high-performance liquid chromatographic system equipped with a UV detector set at 220 nm and an ODS Hypersil column is used. The method met precision, specificity, and accuracy requirements. The hormonal levels determined in one target mare throughout pregnancy were 97.91 to 449.13, 116.47 to 266.02, 74.92 to 235.54, and 84.26 to 300.03 ng/mL, reaching a maximum towards the 25th, 20th, 33rd, and 27th weeks, respectively, for E1, EL, EQ, and 17dEQ. The method was successfully tested by quantitating these estrogens in the plasma from a pregnant mare. Its applicability to the study of estrogen bioavailability and bioequivalence is suggested.     

Reversed-phase liquid chromatographic method for the determination of selected room-temperature ionic liquid cations

The separation of selected 1-alkyl- and 1-aryl-3-methylimidazolium-based room temperature ionic liquid cations has been performed using reversed-phase high-performance liquid chromatography with electrospray ionization mass detection. The RP-HPLC method development started with the selection of a column taking into account especially the resolution of low molecular congeners of the selected group. Mobile phase composition was optimized for peak resolution, sensitivity and high reproducibility of retention values. The results of the method development were applied to the determination of exemplary ionic liquid species present in the medium used in cytotoxicity studies.     

Reversed-phase ion-pair chromatographic separation of selenite and selenate with selenium-specific detection

Tetraalkylammonium salts [R₃RN]⁺X^– (R=R, XCH₃, Cl; C₂H₅, Br; C₃H₇, Br; C₄H₉, H₂PO₄; C₅H₁₁ Br; R, R, XCH₃, C₁₆H₃₃, Br) were investigated as ion-pairing reagents for the reversed-phase ion-pair chromatographic separation of selenite and selenate. Other chromatographic parameters such as composition and pH of the mobile phase and concentration of the ion-pairing reagent were also investigated. The compatibility of the proposed chromatographic procedures with various selenium-specific detectors is discussed. The absolute detection limits were found to be 31 ng Se for selenite and 51 ng Se for selenate (100 l injection) with a solution of 5 mM tetrabutylammonium dihydrogen phosphate in 5050 (v/v) methanol/water as mobile phase at a flow rate of 1 ml/min when a flame atomic absorption spectrometer was used as detector. The HPLC/FAAS system was employed for the determination of selenite in solutions serving as selenium supplement for animals.     

Reversed-phase liquid chromatographic determination of riboflavin in feeds

A method for determination of riboflavin in animal feeds using liquid chromatography (LC) was developed for feed samples fortified with riboflavin at 1 mg/lb or greater (up to 10,000 mg/lb). Feed samples were extracted in 0.1 N HCl with heating on a steam bath for 30 min, followed immediately by mechanical shaking for 30 min. Sample extracts were diluted to target volume with 2% acetic acid and filtered; riboflavin was determined by LC on a reversed-phase C18 column with 2% acetic acid-acetonitrile (85 + 15) mobile phase for separation and fluorescence detection with excitation at 460 nm and emission at 530 nm. The extraction was compared with that of the AOAC Official Method for riboflavin in food and feed premixes. The 2 method extractions were not significantly different from each other at the 95% confidence level. The developed method also had good linearity over 4 orders of magnitude, recovery of 95-99% from spiked feed samples, a limit of detection of riboflavin at 0.00034 microg/mL in solution, a limit of quantitation of 0.023 mg/lb in feed, and good ruggedness.     

Reversed-phase high-performance liquid chromatographic method for the assay of oxytetracycline

The British Pharmacopoeia monograph for oxytetracycline calcium describes an high-performance liquid chromatographic (HPLC) assay which requires packing of the column by the analyst. Presented in this report is an HPLC method for the assay of oxytetracycline which employs a commercially available reversed-phase column and a solvent system which gives improved separation of the antibiotic from common impurities. Results obtained using this method for both bulk and dosage forms of oxytetracycline are in accord with the results of the microbiological assays.     

Reversed-phase high-performance liquid chromatographic method for the quantitative determination of alkylbis(2-benzothiazolylsulfen)amides

An isocratic, reversed-phase HPLC procedure was developed for the simultaneous determination of isopropyl-, tert.-butyl-, tert.-amyl-, cyclohexylbis(2-benzothiazolylsulfen)amides. Quantitation is performed on a C₁₈ bonded-phase column (Separon SGX C₁₈ 5 μm) using N-dicyclohexyl-2-benzothiazolesulfenamide as internal standard followed by UV photodiode-array detection. The precision (n = 7) for all derivatives of alkylbis(2-benzothiazolylsulfen)amides is within 1.5%. Identification of the compounds also in the mixtures was done by NMR spectroscopy.     

Reversed-phase ion-pair liquid chromatography of the angiotensins

The isocratic reversed-phase liquid chromatography of the angiotensins and a number of their synthetic analogues is described. Complete separation of 10 out of 12 peptides was achieved through a solvent optimization strategy with a total analysis time of about 20 min. The retention behavior of the angiotensins studied was described in terms of the hydrophobic contribution of their amino acid residues; there was good correlation between predicted and experimental retention for those peptides that were retained by a common mechanism. However, because ion-pair chromatography was required for good peak symmetry, retention was substantially modulated by the presence of acidic and basic residues. The limit of detection of these peptides was 3-5 pmol by UV absorbance at 214 nm. For those peptides containing a primary amino group the detection limit was improved by two orders of magnitude by fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde/cyanide to the corresponding N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives. The contribution of the CBI ring system to retention was also investigated.     

Reversed-phase ion-pair liquid chromatographic procedure for the simultaneous analysis of S-adenosylmethionine, its metabolites and the natural polyamines

A method using reversed-phase ion-pair liquid chromatography with dual detection was developed for the simultaneous determination of the S-adenosylmethionine (SAM) analogues and the natural polyamines. The separation is obtained with a gradient elution and by adjusting the concentration of octanesulfonic acid used as ion-pairing agent, the ionic strength of the eluent, the pH and the acetonitrile content of the eluents. The SAM analogues are analyzed by UV detection at 254 nm and the polyamines by fluorescence detection after post-column derivatization with o-phthalaldehyde. The method allows the determination of the SAM analogues and the polyamines in one single run by direct injection of tissue extracts. The procedure is applied to the study in rats and in hepatoma tissue culture cells of the biochemical effects of alpha difluoromethylornithine, a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase.     

Reversed-phase ultra-performance liquid chromatographic method development and validation for determination of impurities related to torsemide tablets

A simple RP-ultra-performance LC method was developed and validated for determination of impurities related to torsemide tablets. The rapid method provided adequate separation of all known related impurities and degradation products. Separation was achieved on a Zorbax SB-C18 column (50 x 4.6 mm id, 1.8 microm particle size) with binary gradient elution, and detection was performed at 288 nm. The drug product was subjected to oxidative, hydrolytic, photolytic, and thermal stress conditions to prove the specificity of the proposed method. The linearity and recovery were investigated for known impurities in the range of 0.025 to 1.0%, with respect to the drug concentration in the prepared sample. The linearity of the calibration curve for each of the impurities and torsemide was found to be very good (r2 > 0.999). Relative response factors for each of the known impurities were established by the slope ratio method from the linearity study.     

Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations

A stability indicating, reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of risedronate in pharmaceutical dosage forms. The determination was performed on a BDS C₁₈ analytical column (250 mm × 4.6 mm i.d., 5 μm particle size); the mobile phase consisted of 0.005 M tetrabutylammonium hydroxide and 0.005 M pyrophosphate sodium (pH 7.0) mixed with acetonitrile in a ratio (78:22, v/v) and pumped at a flow rate 1.00 mL min⁻¹. The ultraviolet (UV) detector was operated at 262 nm. The retention times of magnesium ascorbyl phosphate, which was used as internal standard and risedronate were 4.94 and 5.95 min, respectively. The calibration graph was ranged from 2.50 to 20.00 μg mL⁻¹, while detection and quantitation limits were found to be 0.48 and 1.61 μg mL⁻¹, respectively. The intra- and inter-day percentage relative standard deviations, %R.S.D., were less than 5.9%, while the relative percentage error, %E_r, was less than 0.4%. The method was applied to the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.