Application of continuous-flow liquid chromatography/fast-atom bombardment mass spectrometry to the analysis of diagnostic acylcarnitines in human urine

Acylcarnitine profiles of diagnostic value were generated from the equivalent of 0.1 microL of raw urine using the continuous-flow liquid chromatography/fast-atom bombardment mass spectrometry (LC/FAB-MS) interface for sample introduction. Further analysis was accomplished by gradient LC/MS using a commercially available packed fused-silica microbore column.     

High-performance thin layer chromatography method for quantitative determination of four major anthraquinone derivatives in Rheum emodi

A high-performance thin layer chromatographic (HPTLC) method for the rapid and simple quantification of the four major anthraquinone derivatives i.e. physcion, chrysophanol, emodin and chrysophanol glycoside in Rheum emodi is described. HPTLC of anthraquinone derivatives was performed on pre-coated RP-18 F254S HPTLC plates. For achieving good separation, the mobile phase of methanol-water-formic acid (80:19:1, v/v/v) was used. The densitometric determination of anthraquinone derivatives was carried out at 445 nm in reflection/absorption mode. The calibration curves were linear in the range of 20-100 ng for physcion, 80-400 ng for chrysophanol and emodin, and 200-1000 ng for chrysophanol glycoside. The method was found to be reproducible and convenient for quantitative analysis of anthraquinone derivatives in the methanolic extract of rhizomes of R. emodi collected from three different locations of Western Himalaya, India.     

Analysis of saxitoxin in urine by continuous-flow fast-atom bombardment mass spectrometry.

An improved method of saxitoxin analysis in urine using continuous-flow fast-atom bombardment mass spectrometry was developed. Parameters studied were matrix composition, matrix flow, temperature of probe tip, probe-tip design and sample extraction. Optimal detection was obtained using the following matrix composition: 5% glycerol, 0.5% acetic acid, 0.025% sodium dodecylsulfate, 0.1% polyethylene glycol (PEG) 400 and 0.5% PEG 300; probe-tip temperature: (approximately 55 degrees C); flow rate: 5 or 8 microL per min.; probe tip: Olson-Hogge design. The STX standard was detected at 200 pg with signal-to-noise ratio of 11. The percent recovery of saxitoxin from human urine after clean-up on a weak cation exchange column was 75%.     

Direct determination of four fluoroquinolones,enoxacin, norfloxacin,ofloxacin, and ciprofloxacin,in pharmaceuticals and blood serum by HPLC

A rapid, accurate and sensitive method has been developed for the quantitative determination of four fluoroquinolone antimicrobial agents, enoxacin, norfloxacin, ofloxacin and ciprofloxacin, with high in-vitro activity against a wide range of Gram-negative and Gram-positive organisms.A Kromasil 100 C(8) 250 mm x 4 mm, 5 microm analytical column was used with an eluting system consisting of a mixture of CH(3)CN-CH(3)OH-citric acid 0.4 mol L(-1) (7:15:78 %, v/v). Detection was performed with a variable wavelength UV-visible detector at 275 nm resulting in limits of detection: 0.02 ng per 20 microL injection for enoxacin and 0.01 ng for ofloxacin, norfloxacin and ciprofloxacin. Hydrochlorothiazide (HCT) was used as internal standard at a concentration of 2 ng microL(-1). A rectilinear relationship was observed up to 2 ng microL(-1) for enoxacin, 12 ng microL(-1) for ofloxacin, 3 ng microL(-1) for norfloxacin, and 5 ng microL(-1) for ciprofloxacin. Separation was achieved within 10 min. The statistical evaluation of the method was examined by performing intra-day (n=8) and inter-day precision assays (n=8) and was found to be satisfactory with high accuracy and precision. The method was applied to the direct determination of the four fluoroquinolones in human blood serum. Sample pretreatment involved only protein precipitation with acetonitrile. Recovery of analytes in spiked samples was 97+/-6% over the range 0.1-0.5 ng microL(-1).     

Determination of ivermectin B(1a) in animal plasma by liquid chromatography combined with electrospray ionization mass spectrometry

A novel, sensitive and specific method for the quantitative determination of ivermectin B(1a) in animal plasma using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is presented. Abamectin was used as the internal standard. Extraction of the samples was performed with a deproteinization step using acetonitrile. Chromatographic separation was achieved on a Nucleosil ODS 5 microm column, using gradient elution with 0.2% (v/v) acetic acid in water and 0.2% (v/v) acetic acid in acetonitrile. The method was validated according to the requirements defined by the European Community. Calibration curves using plasma fortified between 1 and 100 ng ml(-1) showed a good linear correlation (r > or = 0.9989, goodness-of-fit coefficient < or =8.1%). The trueness at 2 and 25 ng ml(-1) (n = 6) was +4.2 and -17.1%, respectively. The trueness and between-run precision for the analysis of quality control samples at 25 ng ml(-1) was -4.0 and 11.0%, respectively (n = 16). The limit of quantification of the method was 1.0 ng ml(-1), for which the trueness and precision also fell within acceptable limits. Using a signal-to-noise ratio of 3 : 1, the limit of detection was calculated to be 0.2 ng ml(-1). The specificity was demonstrated with respect to ivermectin B(1b).The method was successfully used for the quantitative determination of ivermectin B(1a) in plasma samples from treated bovines, demonstrating the usefulness of the developed method for application in the field of pharmacokinetics.     

Instrumental planar chromatographic method for determination of carbamazepine in human serum

An instrumental planar chromatographic (HPTLC) method for quantification of carbamazepine in human serum was developed using liquid‐liquid extraction with dichloromethane, fluorescence activation with perchloric acid 60%/ethanol/water (1:1:1, v/v) and fluorescence detection. Planar chromatographic separation was performed on precoated silica gel F254 HPTLC plates using a mixture of ethyl acetate/toluene/methanol/acetic acid glacial (5:4:0.5:0.5, v/v) as mobile phase. Densitometric detection was done at 366 nm. The method was validated for linearity, precision and accuracy. Linear calibration curves in the range of 3 and 20 ng/μL showed correlation coefficient of 0.998. The intra‐assay and inter‐assay precision, expressed as the RSD, were in the range of 0.41–1.24% (n = 3) and 2.17–3.17% (n = 9), respectively. The LOD was 0.19 ng, and the LOQ was 0.57 ng. Accuracy, calculated as percentage recovery, was between 98.98 and 101.96%, with a RSD not higher than 1.52%. The method was selective for the active principle tested. In conclusion, the method is useful for quantitative determination of carbamazepine in human serum.     

Simultaneous determination of nitroglycerin and its dinitrate metabolites by capillary gas chromatography with electron-capture detection

A sensitive gas chromatographic-electron-capture detection method for the simultaneous determination of the antianginal drug nitroglycerin (GTN) and its dinitrate metabolites (1,2-GDN and 1,3-GDN) was developed. Human plasma samples (1 ml) spiked with 2,6-dinitrotoluene as the internal standard were extracted once with 10 ml of a methylene chloride-pentane mixture (3:7, v/v). Using this solvent system, less contaminants are extracted into the organic phase from plasma, resulting in cleaner chromatograms and prolonged column life. A break point was observed on the standard curves of GTN and GDNs. The two linear regions for the detectable concentrations of GTN are 0.025-0.3 and 0.3-3 ng/ml and for 1,2-GDN and 1,3-GDN they are 0.1-1 and 1-10 ng/ml. The limits of detection by this method for GTN, 1,2-GDN and 1,3-GDN in plasma are 0.025, 0.1 and 0.1 ng/ml, respectively.     

Simultaneous determination of clozapine, clozapine N-oxide, N-desmethylclozapine, risperidone, and 9-hydroxyrisperidone in plasma by high performance liquid chromatography with ultraviolet detection

A simple high performance liquid chromatography techniques with ultraviolet detection (HPLC–UV) method is described for the simultaneous determination of clozapine (CZP), clozapine N-oxide (CNO), N-desmethylclozapine (NCZ), risperidone (RSP) and 9-hydroxyrisperidone (9-OHRSP) in human plasma. After extraction process, the analytes were separated on a C₁₈ column (150 mm×3.9 mm i.d.) by the mobile phase (methanol–water–dimethylamine, 60:40:0.04 (v:v:v)). Relative recoveries of five analytes were quantitative. The precision and accuracy of intra- and interday assays were all below 8.2% for R.S.D. and 5.6% for RE, respectively. Based on 1 ml of plasma, the limits of detection were 2.0 ng/ml for CZP, 0.2 ng/ml for CZP N-oxide, 1.0 ng/ml for NCZ, 1.0 ng/ml for RSP, and 0.5 ng/ml for 9-OHRSP (S/N=3). The calibration curves were linear (r≥0.988). This method was applied to therapeutic drug monitoring of schizophrenia patients receiving CZP or RSP therapy.     

Identification and quantification of molecular species of diacyl glyceryl ether by reversed-phase high-performance liquid chromatography with refractive index detection and mass spectrometry

We have developed a method to identify and quantify the molecular species of diacyl glyceryl ether (DAGE) using high-performance liquid chromatography (HPLC) equipped with a refractive index detector and an electrospray ionization and time of flight mass spectrometer (LC-RI-MS). An octadecyl silica column with a mixture of acetonitrile and dichloromethane (65:35, v/v) as an eluant was used for the HPLC. When the LC-RI-MS method was applied to a mixture of synthetic DAGEs; 1-O-hexadecyl-2,3-dioleoylglycerol (O-16:0-18:1-18:18:1), 1-O-octadecyl-2,3-dioleoylglycerol (O-18:0-18:1-18:1), 1-O-octadecenyl-2,3-dioleoylglycerol (O-18:1-18:1-18:1), 1-O-octadecyl-2,3-didocodahexaenoylglycerol (O-18:0-22:6-22:6), and 1-O-octadecenyl-2,3-didocosahexaenoylglycerol (O-18:1-22:6-22:6), good separation and quantification were obtained on the refractive index chromatogram. A pseudo-molecular ion [M+NH4]+ and a monoacyl glyceryl ether ion [M-RCO2] + were observed for all synthetic DAGEs on the mass spectrum. It was found that the fatty acids and glyceryl ether in DAGE could be easily identified by these mass spectra. When this LC-RI-MS method was applied to the DAGEs extracted from muscle of Stromateus stellatus, approximately 18 peaks were observed on LC-RI-MS chromatograms and the major molecular species of DAGEs were identified as O-16:0-18:1-18:1.     

A simple and rapid HPLC‐UV method for the determination of retigabine in human plasma

A simple and rapid high‐performance liquid chromatographic method with ultraviolet detection was developed for the quantitative determination of retigabine, known also as ezogabine, in human plasma. The assay uses a simple solid‐phase extraction for sample preparation and direct injection of the extract into the chromatograph. Flupirtine is used as an internal standard. Chromatographic separation is achieved on a C₁₈ Chromolith column (Chromolith Performance, 100 × 4.6 mm i.d.), using as mobile phase water/acetonitrile/methanol (72:18:10 v/v/v) mixed with 0.1% of 85% phosphoric acid. Isocratic elution is conducted at a flow rate of 1.5 mL min⁻¹. The total duration of a chromatographic run is 7 min. Calibration curves are linear over the 25–2000 ng mL⁻¹ concentration range, with a limit of quantitation of 25 ng mL⁻¹. Other performance characteristics include high precision (intra‐ and inter‐day coefficients of variation ≤12.6%) and high accuracy (99.7%–108.7%). The method is suitable for the investigation of concentration–response relationships in patients receiving therapeutic doses of retigabine.     

Determination of some polycyclic aromatic hydrocarbons in filter tar of Turkish cigarettes

A simultaneous determination method for the analysis of benzo[a]pyrene (BaP) and dibenzo[a,h]anthracene (DahA) in the filter tar of Turkish cigarettes has been developed. The method involved (a) the extraction of BaP and DahA with n-hexane from ACN solution in which the cigarette filters were extracted, and then (b) purification of the n-hexane extracts by elution on an XAD-2 column using n-hexane/dichloromethane (9:1, v/v) mixture. Separation and quantitative determination of BaP and DahA in the extracts were carried out by HPLC and fluorescence detection on a C18 RP column. The calculated recoveries for BaP and DahA were found in the range of 90-100% for each extraction and clean-up steps. Analysis of various filter tar of Turkish cigarettes showed that an average of 74.28 ng/filter of BaP and 5.24 ng/filter of DahA were present in Turkish cigarettes.     

Rapid high-performance liquid chromatographic method for the determination of propranolol levels in canine and feline plasma.

A sensitive high-performance liquid chromatographic method that does not require organic extraction has been developed for the determination of propranolol levels in canine and feline plasma. Equal volumes of plasma and a mixture of methanol-acetonitrile-0.1 M sodium hydroxide (3:3:4, v/v/v) were added to a microseparation unit with a 10,000 molecular mass cut-off filter. The ultrafiltrate was analyzed by reversed-phase liquid chromatography with fluorimetric detection. The consistency of the recoveries obtained eliminated the need for an internal standard (coefficients of variation less than 4%). Linear regressions for the standard curves (2.5-100 ng/ml) gave correlation coefficients above 0.9955. The detection limit was 1 ng/ml. The assay retains high sensitivity while eliminating laborious sample preparation.     

High-performance liquid chromatographic analysis of saponin compounds in Bupleurum falcatum

A mixture of saponin compounds (saikosaponin c, a, and d) in the 70% ethanol extract of a powdered sample of Bupleuri radix are analyzed by an Inertsil ODS-3 C(18) column at a flow rate of 1.0 mL/min and detection wavelength of 203 nm. Well resolved chromatograms of saikosaponin c, a, and d are obtained with a gradient elution of acetonitrile-water from 40:60 (v/v) to 50:50 (v/v). The total time required for a single analysis is approximately 20 min. Calibration curves for saikosaponin c, a, and d are linear up to 2.5 mg/mL. The coefficient of variability values for saikosaponins in the extract are below 4%, and the recoveries for saikosaponin c, a, and d are 95.2 +/- 1.1, 96.5 +/- 0.9, and 96.2 +/- 1.0%, respectively. The changes in saikosaponin contents for a two-year growth of Bupleurum falcatum are measured by the established high-performance liquid chromatography method.     

Determination of the pyridinium metabolite derived from haloperidol in brain tissue, plasma and urine by high-performance liquid chromatography with fluorescence detection.

A sensitive and selective method for the determination of the pyridinium metabolite (HPP+) derived from the antipsychotic drug haloperidol (HP) in brain tissue, plasma and urine using high-performance liquid chromatography with fluorescence detection is described. The HPP+ present in biological samples was extracted using a Sep-Pak C18 cartridge. Recoveries of HPP+ ranged from 78 to 90%. Final separation and quantitative estimations of HPP+ were achieved on a C18 reversed-phase column employing a mobile phase of acetonitrile-30 mM ammonium acetate (40:60, v/v) containing 10 mM triethylamine and adjusted to pH 3 with trifluoroacetic acid. The fluorescence detection utilized an excitation wavelength of 304 nm and an emission wavelength of 374 nm. Standard curves were linear in the range of 2.5-100 ng/ml for brain tissue homogenate and plasma samples and 10-500 ng/ml for urine samples. The detection limit of HPP+ was about 1 ng/ml in all biological samples. The concentrations of HPP+ in brain tissue, plasma and urine from HP-treated rats were determined using this method.     

Ultramark 1621 as a reference compound for positive and negative ion fast-atom bombardment high-resolution mass spectrometry

Ultramark 1621, a commercially available mixture of fluorinated phosphazines, was found to bea a useful calibration compound for negative and positive ion fast-atom bombardment (FAB) high-resolution mass spectrometry. Ultramark 1621 worked very well with the most widely used matrices such as glycerol, nitrobenzyl alcohol, and triethanolamine. The negative and positive ion FAB mass spectra of Ultramark include a series of intense peaks extending from 700 to 1900 u.     

Enhancement of mass spectrometric detection of LTC4, LTD4, and LTE4 by derivatization

Several acylating reagents are synthesized and used to introduce quatemary phosphonium or ammonium or ternary sulfonium functions into a simple model of a peptido leukotriene (PLT). One of these reagents was selected for further study with LTE₄, LTD₄, and LTC₄. We demonstrate that acylation of the free amine function of PLTs to produce the 5-triphenylphosphoniumvaleryl-amide (TPPV) derivatives enhances chemical stabilities and significantly increases responses in fast-atom bombardment and continuous-flow liquid secondary ion mass spectrometry (CF-LSIMS) relative to the native PLTs. With high-performance liquid chromatography inlet to CF-LSIMS, we demonstrate the facile detection in selected ion monitoring of the TPPV derivative of 3 pg of LTD₄.     

Quantitative determination of some food dyes using digital processing of images obtained by thin-layer chromatography

A high-performance thin-layer chromatographic method combined with image processing of scanned chromatograms was developed for the determination of some food dyes (tartrazine, azorubine and Sunset Yellow) in different products. Porous silica gel with 3-aminopropyl functional groups attached to the matrix was used as stationary phase and a mixture of isopropanol, diethyl ether and ammonia (2:2:1, v/v/v) formed the mobile phase. Quantitative evaluation was performed using special-purpose software. The linearity of the analytical procedure was sustained by the numerical parameters such as correlation coefficient (0.9952-0.9980) and standard error of determination (0.03-0.20). The limits of detection were found to be within the range of 5.21-9.34 ng/spot, and the limits of quantification between 10.21 and 18.09 ng/spot. Recovery studies performed on two levels of concentration gave values between 96.39 and 102.76%. These results show that the regression approach provides rigorous and realistic detection and quantification limits and as a consequence can be routinely applied to other analytical systems. This method does not require expensive analytical instruments compared with classical densitometry and provides a reliable quantitative evaluation with minimum of time.     

Continuous-flow fast atom bombardment mass spectrometry of oligonucleotides

Although frit-fast atom bombardment (frit-FAB) and continuous-flow FAB mass spectrometry have become standard methods for the analysis of peptides and peptide mixtures, these techniques have not been applied previously to the analysis of oligonucleotides. Mobilephase composition, flow rate, and sample size were optimized for the analysis of oligonucleotides by negative ion frit-FAB mass spectrometry (a type of continuous-flow FAB mass spectrometry). With a mobile phase consisting of methanol/water/triethanolamine (80:20:0.5, v/v/w), flow injection frit-FAB analysis of oligonucleotides showed lower limits of detection compared to standard probe FAB mass spectrometry. For example, in order to obtain a signal-to-noise ratio of 3:1, 38 prnol of d(GTIAAC) were required for frit-FAB mass spectrometry and 62 pmol were required for standard probe FAB mass spectrometry. The largest difference between frit-FAB and standard probe FAB was observed for d(pC)₅, for which the limit of detection by frit-FAB was approximately 11-fold lower than by standard FAB mass spectrometry. Adjustment of the mobile phase to pH 7 with trifluoroacetic acid increased the limit of detection (reduced sensitivity) a minimum of sixfold. Equimolar mixtures of two or three oligonucleotides produced deprotonated molecules in identical relative abundances whether analyzed by frit-FAB or standard probe FAB mass spectrometry. Finally, frit-FAB liquid chromatography mass spectrometry was demonstrated by separating mixtures of oligonucleotides on a β -cyclodextrin high-performance liquid chromatography column with a mobile phase containing methanol, water, and triethanolamine.     

A Rapid and Simple High Pressure Liquid Chromatographic Method for Pharmacokinetic Study of Ciprofloxacin in Human Serum

Abstract

A rapid, accurate and sensitive method has been developed for the quantitative determination of ciprofloxacin, a new second-generation quinolone carboxylic acid antimicrobial agent, with high in vitro activity against a wide range of Gram- negative pathogens and Gram-positive cocci.

A Lichrosorb RP-18 250 x 4.0 mm, 5 μm analytical column was used with an eluting system consisting of a mixture of CH₃CN-CH₃OH-Citric acid 0.4 M (1:3:6 v/v). Detection was performed with a variable wavelength UV-visible detector at 275 nm resulting in a limit of detection of 0.2 ng per 20 μl injection. For the quantitative determination theophylline was used as chromatographic internal standard at a concentration of 1.56 ng/μl. A rectilinear relationship was observed up to 20 ng/μl. Analysis time was less than 6 min. The statistical evaluation of the method was examined performing intra-day (n=8) and inter-day calibration (n=8) and was found to be satisfactory with highly accurate and precise results. The method was applied to the direct determination of ciprofloxacin in human blood serum. Sample pretreatment involved only protein precipitation with acetonitrile. Recovery of ciprofloxacin in spiked samples was 98 ± 4% over the range of 0.5–5 mg/μl.     

Determination of the coumarin derivative cloricromene acid in rabbit plasma and platelets.

Two methods for the determination of cloricromene acid in biological samples are described. Cloricromene acid is a catabolite of cloricromene, a coumarin derivative which is active in the cardiovascular system. After oral administration of cloricromene to a rabbit, plasma and platelets were taken at different times and cloricromene acid was then isolated by solid-phase extraction with Sep-Pak C18 cartridges using acetonitrile-tetrahydrofuran-20% aqueous acetic acid (15:11:74, v/v/v) as eluent. The analyses were performed by reversed-phase high-performance liquid chromatography (RP-HPLC) combined with fluorescence detection with excitation at 310 nm and emission at 390 nm. The limit of quantification by RP-HPLC was about 50 pg. The catabolite in the plasma was identified by continuous-flow fast atom bombardment mass spectrometry (CF-FAB-MS), also used as a complementary means of RP-HPLC determination. The results obtained by RP-HPLC and CF-FAB-MS showed good agreement.