Analysis of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors using liquid chromatography–electrospray mass spectrometry

Employing high-performance liquid chromatography–electrospray mass spectrometry, we describe a new assay for monitoring 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. Incubations were carried out with HMG-CoA reductase (rat liver), HMG-CoA and NADPH, and terminated by the addition of HCl. The reaction product, mevalonolactone, and internal standard, were extracted with ethyl acetate, dissolved in methanol, and analyzed by LC–MS. Using an isocratic mobile phase of 10% acetonitrile and 0.1% formic acid (flow-rate, 0.2 ml/min), the protonated molecules of mevalonolactone at m/z 131 and internal standard, β,β-dimethyl-γ-(hydroxymethyl)-γ-butyrolactone, at m/z 145, were detected using selected ion monitoring. The limit of detection was approximately 6.5 pg, and the limit of quantitation was approximately 16.3 pg. Extraction recovery was >90%. The relative standard deviations for intra- and inter-day assays were approximately 4.1±2.7 and 9.4±3.4%, respectively. Mevalonolactone was examined over a period of 3 days and found to be stable. Using this assay, lovastatin and mevastatin inhibited HMG-CoA reductase activity with IC₅₀ values 0.24±0.02 and 2.16±0.31 μM, respectively. These methods offer some advantages over those reported previously which employ radiolabeled substrate and products, and should be useful in searching for compounds that could lower serum cholesterol or alter cell growth and differentiation.     

Simultaneous determination of 8-hydroxy-2′-deoxyguanosine and 5-methyl-2′-deoxycytidine in DNA sample by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry

8-Hydroxy-2′-deoxyguanosine (8-OHdG) and 5-methyl-2′-deoxycytidine (5-mdC) are utilized as useful biomarkers not only for early diagnosis but also for the detection and assessment of high-risk individuals. In the present study, a sensitive and specific method was developed for simultaneous determination of 8-OHdG and 5-mdC in DNA by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry. The limits of quantification for 8-OHdG and 5-mdC were 80 and 40 pg/ml, respectively. The calibration curves of 8-OHdG and 5-mdC were linear over the concentration range of 0.02–100 ng/ml and the correlation coefficients were higher than 0.9990. The intra-day and inter-day relative standard derivative values were in the range of 0.70–7.47% for 8-OHdG and 1.07–7.06% for 5-mdC, respectively. The recoveries were 93.4–108.5% for 8-OHdG and 87.4–104.9% for 5-mdC, respectively. This method was validated by determination of the background levels of 8-OHdG and 5-mdC in calf thymus DNA, and satisfactory results were obtained.     

Determination of zearalenone and its metabolites α- and β-zearalenol in beer samples by high-performance liquid chromatography–tandem mass spectrometry

A fast, robust and sensitive LC–MS–MS method for the determination of zearalenone (ZON) and its metabolites α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) in beer samples is described. Sample preparation was performed by direct RP-18 solid-phase extraction of undiluted beer samples followed by selective determination of analytes by LC–MS–MS applying an atmospheric pressure chemical ionization (APCI) interface. Using the negative ion mode limits of determination of 0.03–0.06 μg l⁻¹ beer and limits of quantification of 0.07–0.15 μg l⁻¹ beer were achieved, which was distinctly more sensitive than in the positive ion mode. Twenty-three beer samples from different countries, produced from different grains and under different brewing conditions, were investigated by this method, but only in one sample could β-ZOL and ZON be detected. Independently of the type of beer, relative standard deviations between 2.1% and 3.3%, a linear working range of 0.15 μg l⁻¹ to 500 μg l⁻¹ beer and recovery rates around 100% could be achieved when zearalanone (ZAN) was used as internal standard.     

Determination of lycopene, α-carotene and β-carotene in serum by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring

A selected-ion monitoring (SIM) determination of serum lycopene, α-carotene and β-carotene by an atmospheric pressure chemical ionization mass spectrometry (APCI–MS) was developed. A large amount of serum cholesterols disturbed the SIM determination of carotenoids by contaminating the segment of interface with the LC–MS. Therefore, separation of carotenoids from the cholesterols was performed using a mixed solution of methanol and acetonitrile (70:30) as the mobile phase on a C₁₈ column of mightsil ODS-5 (75 mm×4.6 mm I.D.). The SIM determination was carried out by introducing only the peak portions of carotenoids and I.S. (squalene) by means of an auto switching valve. In the positive mode of APCI–MS, lycopene, α-carotene and β-carotene were monitored at m/z 537 and I.S. was monitored at m/z 411. This method was linear for all analytes in the range of 15–150 ng for lycopene, 7–70 ng for α-carotene and 25–50 ng for β-carotene. The detection limit of LC–APCI–MS-SIM for carotenoids was about 3 ng per 1 ml of serum (S/N=3). The repeatabilities, expressed as C.V.s, were 10%, 8.4% and 5.3% for lycopene, α-carotene and β-carotene, respectively. The intermediate precisions, expressed as C.V.s, were 11. 2%, 8.8% and 6.5% for lycopene, α-carotene and β-carotene, respectively.     

Analysis of some metabolites of organic solvents in urine by high-performance liquid chromatography with β-cyclodextrin

Chromatographic separation of the metabolites derived from toluene, ethylbenzene, styrene and xylene was carried out on untreated urine samples from factory workers. The elution sequence was as follows: phenylglyoxilic acid, 3-hydroxy-2-butanone, hippuric acid, o-methylhippuric acid, p-methylhippuric acid, m-methylhippuric acid, p-cresol, m-cresol and o-cresol. The stability constants (K_G) of cresol and methylhippuric acid derivatives were evaluated. The capacity factor (k′), selectivity factor (α) and resolution (R_s) are described with a variety of mobile phases containing β-cyclodextrin (β-CD). The optimum concentration ratio of ethanol–water–acetic acid–β-CD was determined to be 20:80:0.3:1.4%. Under these conditions, k′ values of the five metabolites were 2<k′<6, and all α values were greater than 1.5. Simultaneous determinations of the metabolites were carried out in real urine samples from factory workers using the standard addition method. Validation of the method and the detection limit are described under the optimum conditions attained in this experiment.     

Fast quantification of the urinary marker of oxidative stress 8-hydroxy-2′-deoxyguanosine using solid-phase extraction and high-performance liquid chromatography with triple-stage quadrupole mass detection

Exogenous and endogenous oxidants constantly cause oxidative damage to DNA. Since the reactive oxidants itself are not suitable for analysis, oxidized bases like 8-hydroxy-2′-deoxyguanosine (8OHdG) are used as biomarkers for oxidative stress, either in cellular DNA or as elimination product in urine. A simple, fast and robust analytical procedure is described for urinary 8OHdG as an indicator of oxidative damage in humans. The adduct was purified from human urine by applying a single solid-phase extraction step on LiChrolut EN^®. After evaporation of the eluate, the residue was resolved and an aliquote was injected into a HPLC system with a triple quadrupole mass spectrometer. The limit of detection was 0.2 ng ml⁻¹ (7 fmol absolute) when using one product ion as quantifier and two further product ions as qualifier. The coefficient of variation was 10.1% (n=5 at 2.8 ng ml⁻¹ urine). The sample throughput was about 50 samples a day. Thus, this method is more sensitive and much faster than the common method using HPLC with electrochemical detection. The results of a study with nine volunteers investigated at six time-points each over 5 days are presented. The mean excretion of 8OHdG was 2.1 ng mg⁻¹ creatinine (range 0.17–5.9 ng mg⁻¹ creatinine; 4 of 53 samples were below the LOD). A relatively large intra- (relative SD 66%) and inter-individual (relative SD 71%) variation in urinary 8OHdG excretion rates was found.     

A three-phase liquid chromatographic method for δC analysis of amino acids from biological protein hydrolysates using liquid chromatography–isotope ratio mass spectrometry

We report a three-phase chromatographic method for the separation and analysis of δ¹³C values of underivatized amino acids from biological proteins (keratin, collagen, and casein) using liquid chromatography–isotope ratio mass spectrometry (LC–IRMS). Both precision and accuracy of δ¹³C values for standard amino acid mixtures over the range of approximately 8 to 1320 ng of carbon per amino acid on the column were assessed. The precision of δ¹³C values of amino acids was found to be better at higher concentrations, whereas accuracy improved at lower concentrations. The optimal performance for this method was achieved with between 80 and 660 ng of carbon of each amino acid on the column. At amino acid amounts lower than 20 ng of carbon on the column, precision and accuracy may become compromised. The application of this new three-phase chromatographic technique will allow the analysis of δ¹³C of amino acids to be carried out as a routine method and benefit fields of research such as biomedicine, forensics, ecology, nutrition, and palaeodiet reconstruction in archaeology.     

Screening of chlorpropamide in horse plasma by high-performance liquid chromatography with ultraviolet absorbance detection, and confirmation by gas chromatography–mass spectrometry

A chromatographic method was developed to detect and confirm the presence of chlorpropamide (I) in horse plasma samples, for antidoping control. The plasma sample (1 ml) was extracted with dichloromethane and screened by high-performance liquid chromatography, and confirmation of the drug's presence was accomplished by using gas chromatography–mass spectrometry (GC–MS). The limit of detection was found to be 3.5 ng/ml at a signal-to-noise ratio of three. Derivatization of I with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane allowed for highly stable, accurate and sensitive GC–MS analysis. Plasma samples collected after the administration of diabinese were positive for I (one–five days) in all samples analysed.     

DNA primase associated with 10 S DNA polymerase α from calf thymus

Among multiple subspecies of DNA polymerase α of calf thymus, only 10 S DNA polymerase α had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase α through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase α. These results indicate that the primase is tightly bound to 10 S DNA polymerase α. The RNA polymerizing activity was resistant to α-amanitin, required high concentration of all four ribonucleoside triphosphates (800 μM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase α because it was strongly inhibited by araCTP, resistant to d₂TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.     

Improved purification of the membrane-bound hydrogenase–sulfur-reductase complex from thermophilic archaea using ε-aminocaproic acid-containing chromatography buffers

A hydrogenase–sulfur reductase (SR) complex was purified from membrane preparations of the extremely thermophilic, acidophilic archaeon Acidianus ambivalens using a combination of sucrose density gradient centrifugation and column chromatography (FPLC). All chromatographic steps were performed in the presence of 0.5% ε-aminocaproic acid resulting in the elution of the SR complex as a sharp peak. In contrast, chromatography using buffers without ε-aminocaproic acid, or in the presence of detergents, were not successful. The purified A. ambivalens SR complex consisted of at least four subunits with relative molecular masses of 110 000, 66 000, 39 000 and 29 000, respectively. A similar procedure was applied to purify the membrane-bound hydrogenase from Thermoproteus neutrophilus, a non-related extremely thermophilic but neutrophilic archaeon, which consisted of only two subunits with relative molecular masses of 66 000 and 39 000, respectively.     

Enantiomeric separation of tetrahydroisoquinoline alkaloids by high-performance liquid chromatography with β-cyclodextrin as chiral selector

Tetrahydroisoquinoline alkaloids, which are known to be present not only in plants but also in animals, including mammals, can be considered as condensation products of 2-phenylethylamines (e.g., catecholamines) with aldehydes (e.g., acetaldehyde) or 2-oxo acids (e.g., pyruvic acid). In this study the possibility of separating the optical isomers of several tetrahydroisoquinolines by high-performance liquid chromatography was investigated. For isosalsoline, tetrahydropapaveroline and laudanosoline a good enantiomeric separation could be achieved by applying β-cyclodextrin-bonded silica as stationary phase in connection with various mobile phases. With respect to laudanosoline, the addition of β-cyclodextrin as chiral selector to the mobile phase using a C₁₈ reversed-phase column as stationary phase revealed an even higher resolution when compared with the chiral columns. All tested tetrahydroisoquinolines which could be well separated into enantiomers bear a hydroxyl group at carbon atom 7 as a common structural feature. Those alkaloids substituted with a methoxy group on position 7 instead of a hydroxyl group (e.g., salsolidine) failed to be resolved into their optical isomers. Therefore, the presence of a hydroxyl group on C7 of the aromatic ring seems to be conducive to steric discrimination. However, the separation results for 1-carboxysalsolinol were unsatisfactory although this molecule possesses a 7-hydroxyl group. In this case the existence of a carboxyl group on C1 reduced the chiral recognition and thus the enantiomeric resolution. © 1995 Wiley-Liss, Inc.     

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.     

Identification of progesterone and 5α‐dihydroprogesterone (5α‐DHP) in zebras by two‐dimensional high‐performance liquid chromatography and their dependence on the state of reproduction

Because of its low levels in late pregnancy, the relationship of progesterone to pregnancy maintenance in Equidae is not obvious. This study investigated the levels of progesterone (4‐pregnane‐3,20‐dione; P4) and 5α‐dihydroprogesterone (5α‐DHP) during pregnancy in zebras in relation to reproductive state. Blood samples from female zebras (Equus burchelli, E. zebra hartmannae, E. grevyi) were taken at Dvur Kralove Zoo. Progesterone and 5α‐DHP were separated by high‐performance liquid chromatography techniques and detected by cross‐reacting antibodies. Identification of progestins was achieved by comparing the identity of peaks of the samples with a standard. In E. z. hartmannae progesterone, values reached 50 ng/mL at the beginning of pregnancy and dropped to levels below 1 ng/mL during the second half of pregnancy. In contrast, 5α‐DHP increased up to 123 and 183 ng/mL during late pregnancy in E. z. hartmannae and E. burchelli, respectively. In E. grevyi, 5α‐DHP levels of 368 ng/mL were obtained during pregnancy, whereas progesterone values were similar in pregnant and non‐pregnant individuals. These marked differences in the course of progesterone and 5α‐DHP levels point to the importance of 5α‐DHP for pregnancy maintenance in zebras. Zoo Biol 18:325–333, 1999. © 1999 Wiley‐Liss, Inc.     

Simultaneous determination of urinary free cortisol and 6β-hydroxycortisol by liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry and its application for estimating hepatic CYP3A induction

A reversed-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization tandem mass spectrometry (HPLC–APCI-MS–MS) assay was developed to simultaneously determine monkey urinary free cortisol (C) and 6β-hydroxycortisol (6β-OHC) in 8 min. Urine sample (0.5 ml) containing fludrocortisone acetate (F-C) as the internal standard was extracted with ethyl acetate for 5 min with an extraction efficiency of 90% and 75% for C and 6β-OHC, respectively. A Perkin-Elmer Sciex API 3000 triple quadruple instrument was used for mass spectrometric detection and the column eluent was directed to a heated nebulizer probe. The assay was linear over the range 0.25–10 μM for each analyte. The intra- and inter-day relative standard deviation (RSD) over the entire concentration range for both analytes was less than 10%. Accuracy determined at three concentrations (0.8, 2.0 and 8.0 μM) ranged between 95.5 and 108%. The method described herein is suitable for the rapid and efficient measurement of 6β-OHC/C ratio in Rhesus monkey urine following administration of known hepatic CYP3A inducers and can be used to estimate potential CYP3A induction by drug candidates in the process of early drug development.     

Simultaneous determination of serum retinol and α- and γ-tocopherol levels in type II diabetic patients using high-performance liquid chromatography with fluorescence detection

This paper presents a simple reversed-phase high-performance liquid chromatographic method for the simultaneous determination of retinol, and α- and γ-tocopherols in human serum using a fluorescence detector. For chromatographic separation a binary gradient was used: phase A; acetonitrile–butanol (95:5); phase B; water, at a flow-rate of 1.5 ml/min. Serum retinol, and α- and γ-tocopherol levels were measured in patients with non-insulin-dependent diabetes mellitus. Small sample requirement, good reproducibility and sensitivity make this method useful for the determination of the serum levels of these compounds in patients with diabetes mellitus.     

Quantification of trace levels of β-cyclodextrin and substitution patterns in hydroxypropyl-β-cyclodextrin using high-performance liquid chromatography with polarimetric detection

A high-performance liquid chromatographic (HPLC) method has been developed for separation and determination of components in hydroxypropyl-β-cyclodextrin (HP-β-CD). The method involves separation on an amino-bonded HPLC column using water–acetonitrile as a mobile phase with a polarimetric HPLC detector for quantification. It provides good selectivity and sensitivity and can also be used to compare different sources of HP-β-CD and to measure batch to batch variation. The similarity of the values of molar optical rotation for β-cyclodextrin (β-CD) and HP-β-CD suggests that a polarimetric HPLC detector may be used with a straightforward area normalization method, to quantify the proportion of β-CD in any HP-β-CD sample. Trace amounts of β-CD in HP-β-CD have been measured to a precision of 0.01%. © 1993 Wiley-Liss, Inc.     

Determination of isoprostaglandin F2α type III in human urine by gas chromatography–electronic impact mass spectrometry. Comparison with enzyme immunoassay

F₂-Isoprostanes are stable lipid peroxidation products of arachidonic acid, the quantification of which provides an index of oxidative stress in vivo. We describe a method for analysing isoprostaglandin F_2α type III (15-F_2t-IsoP) in biological fluids. The method involves solid-phase extraction on octadecyl endcapped and aminopropyl cartridges. After conversion to trimethylsilyl ester trimethylsilyl ether derivatives, isoprostaglandin F_2α type III is analysed by mass spectrometry, operated in electronic impact selected ion monitoring mode. We have compared enzyme immunoassay (EIA; Cayman, Ann Arbor, MI, USA) to this method with 30 human urine aliquots following the same extraction procedure in order to determine the agreement between both methods. Isoprostaglandin F_2α type III concentrations determined with gas chromatography–mass spectrometry (GC–MS) did not agree with those determined with EIA. Our results suggest that GC–MS and EIA do not measure the same compounds. As a consequence, comparison of clinical results using GC–MS and EIA should be avoided.     

Rapid and sensitive step gradient assays of glutamate, glycine, taurine and γ-aminobutyric acid by high-performance liquid chromatography–fluorescence detection with o-phthalaldehyde–mercaptoethanol derivatization with an emphasis on microdialysis samples

We developed a rapid step-gradient HPLC method for determination of glutamate, glycine and taurine, and a separate method for determination of γ-aminobutyric acid (GABA) in striatal microdialysates. The amino acids were pre-column derivatized with o-phthalaldehyde–2-mercaptoethanol by using an automated refrigerated autoinjector. Separation of the amino acids was established with a non-porous ODS-II HPLC column, late-eluting substances were washed out with a one-step low-pressure gradient. Concentrations of the amino acids were determined with a fixed-wavelength fluorescence detector. The detection limit for GABA was 80 fmol in a 15 μl sample, detection limits for glutamate, glycine and taurine were not determined because their concentrations in striatal perfusates were far above their detection limits. Total analysis time was less than 12 min, including the wash-out step. The methods described are relatively simple, sensitive, inexpensive, and fast enough to keep up with the microdialysis sampling.     

Comparison of plasma sample purification by manual liquid–liquid extraction, automated 96-well liquid–liquid extraction and automated 96-well solid-phase extraction for analysis by high-performance liquid chromatography with tandem mass spectrometry

Three extraction procedures were developed for the quantitative determination of a carboxylic acid containing analyte (I) in human plasma by high-performance liquid chromatography (HPLC) with negative ion electrospray tandem mass spectrometry (MS–MS). The first procedure was based on the manual liquid–liquid extraction (LLE) of the acidified plasma samples with methyl tert.-butyl ether. The second procedure was based on the automation of the manual LLE procedure using 96-well collection plates and a robotic liquid handling system. The third approach was based on automated solid-phase extraction (SPE) using 96-well SPE plates and a robotic liquid handling system. A lower limit of quantitation of 50 pg/ml was achieved using all three extraction procedures. The total time required to prepare calibration curve standards, aliquot the standards and plasma samples, and process a total of 96 standards and samples by manual LLE was three-times longer than the time required for 96-well SPE or 96-well LLE (4 h, 50 min vs. 1 h, 43 min). Even more importantly, the time the bioanalyst physically spent on the 96-well LLE or 96-well SPE procedure was only a small fraction of the time spent on the manual LLE procedure (<10 min vs. 4 h, 10 min). It should be noted that the 96-well SPE procedure incorporated the two steps of evaporation of the eluates to dryness and subsequent reconstitution of the dried extract. The total time required for the 96-well SPE could be reduced by 50% if the eluates were injected directly, eliminating the drying and reconstitution steps, which is achievable when sensitivity is less of an issue.