Quantitation of biological retinoids by high-pressure liquid chromatography: primary internal standardization using tritiated retinoids

A single method is described for quantitation of 14 retinoids found in biological material. The method consists of reversed-phase HPLC, internal standardization, and carrier extraction procedures with three synthetic retinoids. Primary standardization of HPLC uv detector is achieved using tritiated all-trans-retinoic acid, all-trans-retinol, all-trans-retinyl palmitate, and all-trans-retinyl acetate. Extraction methods are standardized by correlating the uv absorbance of retinoids at 340 nm with radioactivity of tritiated retinoids of known specific activity. Quantitation of 10 pg of tritiated or 5 ng of nonradioactive retinoid per 0.1 g sample in a polarity range from 4-oxo-retinoic acid to retinyl stearate can be achieved in a single, 50-min chromatographic run. A single HPLC pump, a C18 reversed-phase analytical column, a multistep three-solvent gradient, and inexpensive solvents based on methanol, water, and chloroform comprise this cost-effective chromatographic system. Our primary standardization method allows investigators employing different procedures to compare results between laboratories by standardizing the HPLC uv detector with commercially available tritiated retinoids. With this method we were able to quantitate nanomolar amounts of endogenous retinoic acids and retinyl esters, that "HPLC uv only" conditions usually would not detect in the circulation and liver of rats under physiological conditions.     

New trends in sample preparation: on-line microextraction in packed syringe (MEPS) for LC and GC applications Part III: Determination and validation of local anaesthetics in human plasma samples using a cation-exchange sorbent, and MEPS-LC-MS-MS

The need for on-line sample preparation for high-throughput applications in bioanalysis has increased during the past decade. In this paper a robust and on-line sample preparation technique, micro extraction in packed syringe (MEPS) has been developed and validated. The method is a miniaturized, fully automated, solid-phase extraction (SPE) technique that can be connected on-line to GC or LC without any modification of the chromatographs. The performance of MEPS as sample preparation method is illustrated by the determination of local anaesthetics in human plasma samples on-line with high performance liquid chromatography (HPLC) and tandem mass spectrometry. The sampling sorbent was 1mg silica based benzenesulphonic acid cation exchanger that was inserted in a 250 microl syringe. Ropicavine and two of its metabolites (PPX and 3-OH-ropivacine), lidocaine and bupivacine were used as model substances. The accuracy values of quality control samples (QC) were between 95% and 109%, and precision (relative standard deviation, R.S.D.) had a maximum deviation of 9% for the analytes.     

Chromatographic analysis of endogenous retinoids in tissues and serum

We present a reliable, highly sensitive, and versatile method for the simultaneous determination of endogenous polar (acidic) and apolar (retinol, retinal, and retinyl esters) retinoids in various biological matrices. Following a single liquid extraction of retinoids from tissues or plasma with isopropanol, polar retinoids are separated from apolar retinoids and neutral lipids via automated solid-phase extraction using an aminopropyl phase. After vacuum concentration to dryness and reconstitution of the residue in appropriate solvents, the obtained fractions are injected onto two different high-performance liquid chromatography (HPLC)-systems. Polar retinoids are analyzed on a RP18 column (2.1mm ID) using a buffered gradient composed of methanol and water and on-column-focusing large-volume injection. Apolar retinoids are separated on a normal-bore RP18 column using a nonaqueous gradient composed of acetonitrile, chloroform, and methanol. Both HPLC systems are coupled with UV detection, and retinoids are quantitated against appropriate internal standards. The method was validated with regard to recovery, precision, robustness, selectivity, and analyte stability. Using 400 microl serum or 200mg tissue, the limits of detection for all-trans-retinoic acid were 0.15ng/ml or 0.3ng/g, respectively. The corresponding values for retinol were 1.2ng/ml or 2.4ng/g, respectively. This method was successfully applied to mouse, rat, and human tissue and serum samples.     

High-throughput solid-phase extraction for the determination of cimetidine in human plasma

For the implementation and validation of an automated `high-throughput' solid-phase extraction (SPE) system, using microtiter solid-phase technology and a pipetting robot, a SPE method previously validated manually for cimetidine in human plasma was adapted. Sample cleanup was performed by means of SPE using Microlute extraction plates in the 96-well format, each well filled with 50 mg of Varian C₁₈ sorbent. Separation was performed by reversed-phase high-performance liquid chromatography (HPLC) with UV detection at 234 nm. The validated calibration range was from 0.100 to 5.00 mg/l, with an inaccuracy and imprecision below 20% at all concentration levels. Validation results on linearity, specificity, precision, accuracy and stability are shown and are found to be adequate. Cross-check analysis of samples from a clinical trial showed that there is a good correlation between results obtained by the automated method and results obtained by the manual method. The average sample preparation time for a technician decreased from approximately 4 min per sample to 0.6 min. A sample throughput of at least 160 samples per day can be achieved, the HPLC analysis time being the rate-limiting step.     

Automated method for determination of glutardialdehyde residues in flexible endoscopes after disinfection

Glutardialdehyde (GDA) is the most commonly used disinfectant for flexible endoscopes. After inappropriate rinsing of endoscopes residual GDA in the narrow endoscope channels may lead to toxic effects in patients. Common methods for determination of aldehydes in water involve derivatization with 2,4-dinitrophenylhydrazine (DNPH), liquid-liquid or solid-phase extraction and HPLC determination. Since derivatization and extraction is both time-consuming and labor-intensive only a small number of samples can be measured. Thus, we developed a fully automated method which includes a conventional HPLC system, a programmable autosampler, and UV detection. After GDA derivatization using DNPH the samples remain in the aqueous phase and no preconcentration of the analyte is necessary. The samples are automatically derivatized through the autosampler. While derivatization in one sample takes place the previous sample is injected and measured by HPLC. Our method is well suited for screening residual GDA in endoscopes as it is both time- and labor-saving.     

Simultaneous analysis of retinol,all-trans- and 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in plasma by liquid chromatography using on-column concentration after single-phase fluid extraction

A reversed-phase high-performance liquid chromatographic method for the simultaneous analysis of retinol, all-trans-retinoic acid, 13-cis-retinoic acid and 13-cis-4-oxoretinoic acid in human plasma and cell culture medium is described. Sample preparation involves precipitation of proteins and extraction of retinoids with 60% acetonitrile. After centrifugation, the acetonitrile content of the supernatant is reduced to 45%, allowing on-column concentration of analytes. Injection volumes up to 2.0 ml (equivalent to 0.525 ml of sample) can be used without compromising chromatographic resolution of all-trans-retinoic acid and 13-cis-retinoic acid. Retinoids were stable in this extract and showed no isomerization when stored in the dark in a cooled autosampler, allowing automated analysis of large series of samples. Recoveries from spiked plasma samples were between 95 and 103%. Although no internal standard was used, the inter-assay precision for all retinoids was better than 6% and 4% at concentrations of 30 nM and 100 nM, respectively. The method is a valuable tool for the study of cellular metabolism of all-trans-retinoic acid, as polar metabolites of this compound can be detected with high sensitivity in cell culture media.     

13-cis-retinoic acid is an endogenous compound in human serum

The occurrence of 13-cis-retinoic acid as an endogenous component in human serum has been confirmed by cochromatography with standards in both normal-phase and reverse-phase high-performance liquid chromatographic (HPLC) system, by the lambda max of its UV spectrum recorded simultaneously with the HPLC run, and by chromatography of its methyl derivative. The method using solid-phase extraction followed by a gradient reverse-phase HPLC procedure with an internal standard and sensitive UV detector, provides an efficient and sensitive technique for the separation and quantification of serum 13-cis- and all-trans-retinoic acid. Serum levels of 13-cis- and all-trans-retinoic acid in 26 fasting volunteers ranged from 1.0 to 2.2 ng/ml (mean +/- SEM = 1.4 +/- 0.3 ng/ml) and from 1.1 to 1.9 ng/ml (mean +/- SEM = 1.4 +/- 0.2 ng/ml), respectively. The levels determined by a liquid-liquid double-phase extraction method were 90% higher in both 13-cis- and all- trans-retinoic acid than those from a solid-phase extraction. Human small intestine can isomerize all-trans-retinoic acid. 13-cis-Retinoic acid is the predominant cis isomer after incubation of intestinal mucosa homogenates with all-trans-retinoic acid. Moreover, the concentration of retinoic acid in serum is related to diet in that the level of total retinoic acid was 36% higher (n = 10) 2 h after a nonstandard breakfast than in fasting subjects.     

High-performance liquid chromatographic method for the simultaneous determination of the camptothecin derivative irinotecan hydrochloride, CPT-11, and its metabolites SN-38 and SN-38 glucuronide in rat plasma with a fully automated on-line solid-phase extraction system, PROSPEKT

We established a high-performance liquid chromatography (HPLC) method for the simultaneous determination of the camptothecin (CPT) derivative, irinotecan hydrochloride (CPT-11) and its metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38) and SN-38 glucuronide (SN-38G) in rat plasma with a fully automated on-line solid-phase extraction system, PROSPEKT. Plasma samples were pretreated with 0.146 M H₃PO₄ to inactivate carboxylesterase and β-glucuronidase in rat plasma, and added with the internal standard solution (0.146 M H₃PO₄ containing 1 μg/ml CPT) and then analyzed. The method was validated for CPT-11 (5 to 25 000 ng/ml), SN-38 (5 to 2500 ng/ml) and SN-38G (2.5 to 500 ng/ml). This method enabled the determination of many samples within a relatively short time with easy sample preparation. It also had four advantages compared with conventional determination methods, i.e. automation of a complicated sample preparation, time-saving by the simultaneous determination of three compounds, the direct determination of SN-38G, and the small amount of plasma required for the determination.     

Reversed-phase gradient high-performance liquid chromatographic procedure for simultaneous analysis of very polar to nonpolar retinoids, carotenoids and tocopherols in animal and plant samples

A reversed-phase gradient high-performance liquid chromatographic (HPLC) procedure, which utilizes gradient elution and detection by a photodiode-array detector, has been developed to analyze simultaneously very polar retinoids, such as 4-oxo-retinoyl-β-glucuronide, retinoyl β-glucuronide and 4-oxo-retinoic acid; polar retinoids, such as retinoic acid and retinol; nonpolar retinoids, such as retinyl esters; along with xanthophylls, monohydroxy carotenoids, hydrocarbon carotenoids, and tocopherols. The procedure has been applied to the simultaneous analysis of retinoids, carotenoids, and tocopherols present in human serum and liver, rat serum and tissues, and for carotenoids in a number of fruits and vegetables. Bilirubin present in human serum can also be simultaneously analyzed. By this gradient HPLC procedure, 3,4-didehydroretinyl ester (vitamin A₂ ester) has been identified as a minor constituent in a human liver sample. Lycopene was identified as a major carotenoid in one specimen of papaya fruit, and 5,6,5′,6′-diepoxy-β-carotene was characterized as a major carotenoid in one specimen of mango fruit.     

Automated analysis of the pyridinium crosslinks of collagen in tissue and urine using solid-phase extraction and reversed-phase high-performance liquid chromatography.

A fully automated method for assaying the collagen crosslinking amino acids, pyridinoline and deoxypyridinoline, in human urine samples or tissue hydrolysates is described. Samples were processed using a Gilson ASPEC system with solid-phase extraction of the crosslinks on columns containing 100 mg of microgranular cellulose. Introduction of an additional solvent step during sample preparation allowed direct analysis by reversed-phase HPLC and elimination of the drying step used previously in a manual method. Use of a synthetic pyridinoline derivative as internal standard enabled accurate quantification of the crosslinks by correcting for recoveries through the whole assay. Samples were analyzed in sequential mode with a total assay time of 30 min. The automated assay showed close correlation with the manual method for both free and total crosslink determinations in human urine (r > 0.97). Reproducibility was improved, as seen from replicate analyses of human urine (CV < 3% for automated pyridinoline measurement compared with 8-12% previously observed for the manual method). Crosslink excretion is the most useful marker of collagen degradation in metabolic bone diseases and arthritic disorders. The automated assay which has been developed is rapid, convenient, and reliable and will greatly facilitate the monitoring of urinary collagen crosslinks and their tissue levels in clinical investigations.     

Molecularly imprinted polymer cartridges coupled on-line with high performance liquid chromatography for simple and rapid analysis of dextromethorphan in human plasma samples

In this paper, a novel method is described for automated determination of dextromethorphan in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as a sample clean-up technique combined with high performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and dextromethorphan as template molecule. These imprinted polymers were used as solid-phase extraction sorbent for the extraction of dextromethorphan from human plasma samples. Various parameters affecting the extraction efficiency of the MIP cartridges were evaluated. The high selectivity of the sorbent coupled to the high performance liquid chromatographic system permitted a simple and rapid analysis of this drug in plasma samples with limits of detection (LOD) and quantification (LOQ) of 0.12 ng/mL and 0.35 ng/mL, respectively. The MIP selectivity was evaluated by analyzing of the dextromethorphan in presence of several substances with similar molecular structures and properties. Results from the HPLC analyses showed that the recoveries of dextromethorphan using MIP cartridges from human plasma samples in the range of 1-50 ng/mL were higher than 87%.     

On-line extraction using an alkyl-diol silica precolumn for racemic citalopram and its metabolites in plasma: Results compared with solid-phase extraction methodology

Sample preparation is usually the most critical and time consuming step when using HPLC for drug analysis in biological matrixes. Sample extracts have to be clean considering both chromatographic interferences and column maintenance. To meet some of these criteria a fully automated on-line extraction (OLE) analysis method was developed for the antidepressant drug citalopram and its two demethylated metabolites, using an RP-C4-ADS extraction column. A comparison between the new OLE method and an off-line solid-phase extraction method showed that the two methodologies were equal in analytical precision but that the OLE method was faster and therefore superior in sample capacity per day.     

High-throughput bioanalytical method using automated sample preparation and liquid chromatography-atmospheric pressure ionspray mass spectrometry for quantitative determination of glybenclamide in human serum

A liquid chromatographic-mass spectrometric (LC-MS) method with rapid automated sample preparation was developed and validated for determination of glybenclamide in human serum. Glybenclamide and its deuterated labelled internal standard were extracted from human serum samples by automated solid-phase extraction. The extract was injected into the LC-MS system for analysis. Glybenclamide and its internal standard were measured in multiple ion monitoring mode. The method was validated over a range of 10-1000 ng/ml with good accuracy and precision and was applicable for pharmacokinetic studies.     

High-performance liquid chromatographic method for an automated determination of local anaesthetics in human plasma

A method is described that allows the rapid and precise determination of the local anaesthetics bupivacaine and etidocaine from biological fluids. This method uses a fully automated system with solid-phase extraction in combination with a column-switching technique. Both sample extraction on a LiChrocart pre-column and elution onto the analytical LiChrospher column, were performed automatically and concomitantly using conventional HPLC equipment in conjunction with an OSP-2 on-line sample preparator from Merck combined with UV detection. Recoveries were found to be 96.7 and 96.4% for 2 μg/ml bupivacaine and etidocaine, respectively. Lower limits of quantification were found to be 0.05 μg/ml plasma for both of the compounds.     

Development of a method for sample preparation for subsequent identification and measurement of 1,2,3,4-tetrahydroisoquinolines and other potentially neurotoxic compounds by high-performance liquid chromatography with ultraviolet and fluorescence detection in blood plasma of Parkinson’s disease patients

We developed sample preparation methods for the detection of various biogenic phenylethylamine derivatives such as 3,4-dihydroxyphenylalanine, and their cyclisation products with aldehydes, i.e., 1,2,3,4-tetrahydroisoquinoline derivatives in blood samples. 1,2,3,4-Tetrahydroisoquinolines are considered to play an essential role as neurotoxic compounds in the pathomechanism of Parkinson’s disease. We used reversed-phase high-performance liquid chromatography with ultraviolet and fluorescence detection for separation and identification. Ultrafiltration, protein precipitation and solid-phase extraction were investigated for purification of blood samples and enrichment of various compounds with a wide range of hydrophilicity and hydrophobicity. Protein precipitation by methanol and perchloric acid is a fast method to separate the analytes from the plasma matrix. A higher yield of the analytes is attained with prior addition of an alkylsulfonic acid giving a fine-grained precipitate. With the addition of ion pairing compounds into the sample it is possible to enrich not only lipophilic compounds such as norharman, tryptamine and melatonin, but also hydrophilic ones such as 3,4-dihydroxyphenylalanine by reversed-phase solid-phase extraction. Ultrafiltration is not useful as a screening method.     

Analysis of olanzapine in human plasma utilizing reversed-phase high-performance liquid chromatography with electrochemical detection

A sensitive reversed-phase HPLC method for the analysis of olanzapine in human plasma is described. Isolation of olanzapine from plasma was accomplished by solid-phase extraction utilizing an ion-exchange/reversed-phase cartridge designed for basic drug extraction. The drug was subsequently separated by reversed-phase HPLC and monitored by electrochemical detection (ED). Electrochemical analysis was used to detect olanzapine due to its uniquely low oxidative potential. Ascorbic acid was added to prevent oxidation during extraction. The limit of quantitation for the assay was established at 0.25 ng/ml utilizing a 1-ml human plasma sample. The average inter-day accuracy was 96.6% with a average precision (%C.V.) of 3.22% over the concentration range of 0.25 to 100 ng/ml. This method was applied to human plasma samples from human clinical trials with olanzapine. The HPLC-ED method compared favorably with a negative chemical ionization GC-MS method previously utilized for analysis of olanzapine in human plasma.     

Automated purification of synthetic oligonucleotides

We have automated the trityl-on purification of oligonucleotides by use of an XYZ axis robotic solid-phase extraction system. This greatly decreased the preparation time required for oligonucleotide purification. After about 15 min for set up of the samples and instrument, the oligonucleotides are automatically purified with a 15-min run time per sample. Thus, for example, the purification of 15 oligonucleotides requires only about 15 min of preparation time and 4 h of machine time. Yields and purity are equivalent to manual methods.     

Automated analysis of urinary catecholamines by high-performance liquid chromatography and on-line sample pretreatment

A simple and automated solid-phase extraction for the selective and quantitative HPLC analysis of free catecholamines in urine is described. The urinary catecholamines react with diphenylboric acid, giving a complex at pH 8.5 which is strongly retained on a PLRP-S cartridge; elution is accomplished with the same mobile phase used for HPLC analysis. Separation is performed by ion-pair reversed-phase HPLC, with sodium heptanesulphate as counter-ion, and a totally end-capped C₁₈ analytical column. Quantitation is achieved with an electrochemical detector. A Spark Holland Prospekt system controls the on-line solid-phase extraction, preconcentration and direct elution to the LC column. Chromatography run-time is 10 min and the total time to process one urine sample is ca. 12 min.     

Inhibition of liver microsomal lipid peroxidation by 13-cis-retinoic acid

The effects of 13-cis-retinoic acid on iron/ascorbate-dependent lipid peroxidation were investigated with rat liver microsomes. 13-cis-retinoic acid effectively inhibited malondialdehyde generation and molecular oxygen consumption associated with lipid peroxidation. Under the conditions employed, inhibition was complete at concentrations as low as 25 microM and the IC50 was 10 microM. Evidence for concomitant retinoid oxidation by microsomal unsaturated fatty acid-derived peroxyl radicals was demonstrated by detection of several retinoid-derived metabolites, including 5,8-oxy-13-cis-retinoic acid, generated during lipid peroxidation. The data indicate that 13-cis-retinoic acid inhibits lipid peroxidation by scavenging lipid peroxyl radicals with its conjugated polyene system. Its antioxidant properties may contribute to the pharmacological activities of this and related retinoids.     

Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction

We improved our previous analytical method to measure phthalate metabolites in urine as biomarkers for phthalate exposure by automating the solid-phase extraction (SPE) procedure and expanding the analytical capability to quantify four additional metabolites: phthalic acid, mono-3-carboxypropyl phthalate, mono-isobutyl phthalate (miBP), and monomethyl isophthalate. The method, which involves automated SPE followed by isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS), allows for the quantitative measurement of 15 phthalate metabolites in urine with detection limits in the low ng/ml range. SPE automation allowed for the unattended sequential extraction of up to 100 samples at a time, and resulted in an increased sample throughput, lower solvent use, and better reproducibility than the manual SPE. Furthermore, the modified method permitted for the first time, the separation and quantification of mono-n-butyl phthalate (mBP) and its structural isomer miBP. The method was validated on spiked pooled urine samples and on pooled urine samples from persons with no known exposure to phthalates.