Sensitive assay for verapamil in plasma using gas-liquid chromatography with nitrogen-phosphorus detection

A sensitive gas-chromatographic method for quantitative analysis of verapamil in human plasma is described. The method involves a single extraction procedure, followed by separation on a capillary column and detection with a nitrogen-phosphorus detector. The detection limit, based upon an assayed plasma volume of 0.5 ml, is 2 ng/ml. The standard curve is linear in the concentration range of 2 to 1000 ng/ml. The recovery of verapamil by pentane-isopropanol extraction was found to be 95%. Zipeprol is used as the internal standard. No interference from drugs needed for the associated cancer therapy has been found. Serum verapamil concentrations are determined by this method in fourteen cancer patients undergoing treatment with adriamycin.     

A simultaneous assay system for N-acetylgalactosamine and N-acetylgalactosaminitol using gas chromatography/mass spectrometry

A procedure is described, using gas chromatography/mass spectrometry to identify and quantitate various sugar alditol acetates, which will simultaneously quantitate N-acetylgalactosamine and N-acetylgalactosaminitol. N-acetylgalactosamine is distinguished from N-acetylgalactosaminitol by reduction with sodium borodeuteride and determining relative intensities of the ion pairs $\text{m}\text{e}$ 84, 85; 102, 103; and 144, 145. The method is adequate for determining amounts of these sugars in the range of 40–1000 nmol.     

Ultrasensitive assay for three polyphenols (catechin,quercetin and resveratrol) and their conjugates in biological fluids utilizing gas chromatography with mass selective detection

The concentrations of three polyphenols ((+)-catechin, quercetin and trans-resveratrol) in blood serum, plasma and urine, as well as whole blood, have been measured after their oral and intragastric administration, respectively, to humans and rats. The method developed for this purpose utilized ethyl acetate extraction of 100 μl samples and their derivatization with bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by gas-chromatographic analysis on a DB-5 column followed by mass selective detection employing two target ions and one qualifier ion for each compound. Total run time was 17 min with excellent resolution and linearity. The limits of detection (LOD) and quantitation (LOQ) were an order of magnitude less than for any previously published method, being 0.01 μg/l and 0.1 μg/l, respectively, for all compounds. Recovery at 1 μg/l and 10 μg/l was >80% in all instances but one, and was >90% in 50%. Imprecision was acceptable at 0.25 and 1.0 μg/l, concentrations below the LOQ of previous methods. Aglycones released from conjugates after hydrolysis were easily measurable. Optimal conditions for hydrolysis were established. After oral administration of the three polyphenols to humans, their conjugates vastly exceeded the concentrations of the aglycones in both plasma and urine. Concentrations peaked within 0.5–1.0 h in plasma and within 8 h in urine. During the first 24 h, 5.1% of the (+)-catechin and 24.6% of the trans-resveratrol given were recovered in the urine (free plus conjugated). This method can be proposed as the method of choice to assay these polyphenols and their conjugates in biological fluids.     

Confirmation of cannabinoids in meconium using two-dimensional gas chromatography with mass spectrometry detection

Meconium has become the specimen of choice for determining fetal exposure to drugs of abuse, but its physical complexity can cause interferences from matrix effects. A new method to determine 9-carboxy-11-nor-Delta(9)-THC (9-THCA) and 11-hydroxy-Delta(9)-THC (11-OH-THC) using two-dimensional (2D) GC-MS was developed to reduce interferences and carryover. The method was validated using 70 spiked samples prepared in drug-free meconium and 46 residual patient specimens that were confirmed to contain cannabinoids. Ten patient specimens that failed to confirm due to interferences using the previous GC-MS method were analyzed using the new 2D method and 9-THCA was quantitated in all ten samples. The 2D GC-MS method improved chromatography which significantly reduced interferences and carryover when compared to the previous GC-MS method.     

Sensitive assay of trimethylamine N-oxide in liver microsomes by headspace gas chromatography with flame thermionic detection

To compare the trimethylamine N-oxygenase activity of liver microsomes from house musk shrew (Suncus murinus) and rat, a sensitive method for the quantitation of trimethylamine (TMA) N-oxide was developed using gas chromatography with flame thermionic detection. The limit of quantification was 0.5 μM and the calibration curve was linear at least up to 5 μM in incubations containing liver microsomal preparations from Suncus. The intra-day RSD values ranged from 10.4 to 12.8 at 0.5 μM and from 3.5 to 6.7 at 5 μM. The inter-day RSD values were 11.6 and 6.5 at 0.5 and 5 μM, respectively. This method provides a sensitive assay for TMA N-oxygenase activity in liver microsomes. Using this method we found that Suncus was capable of N-oxidizing trimethylamine at a very slow rate.     

An assay for dihydroorotase using high-performance liquid chromatography with radioactivity detection

An assay for measuring dihydroorotase activity was devised. Radiolabeled substrate and product were separated by high-performance liquid chromatography using a reverse-phase column with ion-pairing, and the radioactivity was quantitated by flow detection.     

Sensitive and selective viral DNA detection assay via microbead-based rolling circle amplification

We report a sensitive and efficient magnetic bead-based assay for viral DNA identification using isothermal amplification of a reporting probe.     

Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection

When measuring fentanyl and midazolam simultaneously in the same plasma sample with standard high-performance liquid chromatography–ultraviolet (HPLC–UV) detection, overlap of the fentanyl peak by the midazolam peak occurs, which makes fentanyl determination impossible. We tested the hypothesis that by acidifying the methanol mobile phase with 0.02% perchloric acid, 70%, it would be possible to separate both peaks. The UV detector was set at 200 nm. Calibration curves for fentanyl (range 0–2000 pg/ml) and midazolam (range 0–400 ng/ml) were linear (r>0.99). The detection limits were 200 pg/ml (fentanyl) and 10 ng/ml (midazolam). Precision and accuracy for intra- and inter-assay variability as well as in-line validation with quality control samples (QCS) were acceptable (< 15 and 20%, respectively), except for fentanyl QCS of 200 pg/ml (17.8% precision). Although less sensitive than gas chromatography–mass spectrometry (GC–MS), reliable measurements of fentanyl, simultaneously with midazolam, can be performed with this HPLC–UV system.     

Enantioselective determination of isradipine in human serum using chiral stationary phase liquid chromatography and gas chromatography with nitrogen selective detection

rac-Isradipine is a dihydropyridine type calcium antagonist. Its calcium entry blocking effect is due primarily to the (+)-(S)-enantiomer. This study describes a sensitive enantioselective method for the determination of isradipine in human serum. Following alkaline extraction into hexane, the enantiomers of isradipine are separated quantitatively by high-performance liquid chromatography on a Chiralcel OJ column at 39°C. The collected fractions were evaporated and assayed using capillary gas chromatography on a HP 50+ column with nitrogen selective detection. Using 2.0 ml of serum, 0.7 nmol/1 (0.26 ng/ml) of each enantiomer could be determined with acceptable precision. The method has successfully been used to measure (+)-(S)- and (−)-(R)-isradipine concentrations in samples from volunteers after intravenous and oral administration of isradipine. Chirality 10:808–812, 1998. © 1998 Wiley-Liss, Inc.     

Sensitive and selective method for the determination of chlormezanone in plasma by electron-capture gas chromatography

A sensitive and selective determination method of chlormezanone in plasma has been divised. Chlormezanone in plasma was extracted with toluene at pH 4.5, and converted into p-chlorobenzaldehyde in 0.1 N NaOH. Using p-bromobenzaldehyde as an internal standard, the hydrolysis product and the internal standard were extracted with n-hexane, and the extract was concentrated in vacuo in the presence of isoamyl alcohol to prepare the sample solution. The sample solution was submitted to electron-capture gas chromatography. Chlormezanone was determined by use of the peak height ratio of p-chlorobenzaldehyde against the internal standard. The method was utilized successfully for pharmacokinetic studies of chlormezanone in plasma.     

Analysis of native and oxidized low-density lipoprotein oxysterols using gas chromatography—mass spectrometry with selective ion monitoring

SUMMARY

Cholesterol oxidation products have been demonstrated to possess a wide variety of biological properties and have been implicated in playing an important role in the development of atherosclerosis. We have developed an analytical method using capillary gas chromatography-mass spectrometry (GC-MS) for the analysis of cholesterol oxidation products in low-density lipoprotein (LDL). The method uses programmed multiple selected ion monitoring (SIM), providing enhanced sensitivity and accuracy of peak detection over full-scan mass spectra. The major oxidation products of cholesterol in oxidized LDL were identified as 7β-hydroxy-cholesterol and 7-keto-cholesterol. Minor products included 4β-hydroxy-cholesterol, 6β-hydroxy-cholesterol and cholesterol-5α,6α-epoxide. Native LDL contains 7-lathosterol, which is a biosynthetic precursor of cholesterol, as well as low levels of 7β-hydroxy-cholesterol and 7-keto-cholesterol. 7-Lathosterol was not detected in oxidized LDL. A time course oxidation of native LDL with 8 μM CuCl₂ demonstrated a rapid increase in 7β-hydroxy-cholesterol and 7-keto-cholesterol over the first 4 h. Cholesterol—5α,6α-epoxide, and β4-hydroxy- and 6β-hydroxy-cholesterol levels increased gradually, while 7-lathosterol decreased over the same period. This method was used to measure the levels of 7-lathosterol and cholesterol oxides in the LDL of 20 healthy subjects in order to establish the mean concentration and a reference range. This method can be used for the characterization and quantitation of oxysterols in native and oxidized LDL and may afford an additional index of oxidative modification of plasma lipoproteins.     

An assay for phentolamine using high performance liquid chromatography with electrochemical detection

A new method is presented for the detection of phentolamine by high performance liquid chromatography with electrochemical detection. The electrochemical detector was used in the oxidative mode at +900 mV potential versus Ag/AgCl reference. The on-column detection limit for phentolamine using this method was 3 ng, and detector response was linear for 3-1000 ng injected on column. The coefficient of variation for replicate injections was 2.4%. The measurement of phentolamine in biological samples was accomplished using yohimbime as the internal standard; retention time for yohimbine was 3.0 min while phentolamine eluted at 4.75 min. Biological samples were buffered to pH 9.2 and extracted with diethyl ether, followed by back extraction into 0.1 N HCl. The extraction efficiency for this method was 99.4% for phentolamine in serum and 59.3% in liver tissue. The detection limit for phentolamine was 5 ng/ml for 1.0-ml serum samples, and was 10 ng/ml for 1.0-ml liver homogenate samples. The disappearance of phentolamine from serum and liver after administration of a single ip dose of phentolamine to mice was determined using this method. Absorption from the ip route was rapid, with peak phentolamine concentrations achieved in 15 min or less. The elimination half-life of phentolamine in serum was approximately 50 min and was paralleled by disappearance of phentolamine in the liver.     

Determination of cyclizine and norcyclizine in plasma and urine using gas—liquid chromatography with nitrogen selective detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-a]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-a][1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether—methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 ± 5.4% (S.D.) and 90.3 ± 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration—time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Enzyme assay for protein kinase using micellar electrokinetic chromatography with laser-induced fluorescence detection

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection has been developed for a protein kinase assay. This protein kinase assay could readily determine the phosphorylation activity of substrate peptide kemptide using cAMP-dependent protein kinase (PKA) as a model enzyme. Kemptide and phosphorylated kemptide could be reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent for LIF detection by directly adding NBD-F into the PKA enzymatic reaction mixture. These derivatives of substrate and product were separated and detected within the analysis time of 5 min by micellar electrokinetic mode using a mixture of sodium dodecylsulfate and methanol as a running buffer. Good linearity of the peak response of the phosphorylated kemptide was obtained over the range of 1-20 mU/tube of PKA in the assay. The relative standard deviation of the peak areas of the phosphorylated kemptide using 2, 5 and 10 mU/tube of PKA were calculated to <10.4%, indicating that the assay was reproducible. Also, IC(50) values of six PKA inhibitors, the K(i) value and the inhibition pattern of one inhibitor, which were calculated to estimate by the variation of the peak area of the phosphorylated kemptide using 5 mU/tube of PKA, were consistent with the published data. The sensitivity of the assay was higher than that of enzyme-linked immunosorbent assay (ELISA) for PKA phosphorylation activity, as IC(50) values, K(i) value, and the inhibition mechanism of inhibitors could be estimated using one-tenth amounts of PKA, compared with that of ELISA. The MEKC-LIF is expected to be very useful for protein kinase assay and its application to the estimation of inhibitors because this method does not entail experimentally troublesome procedures such as the preparation of antibody or fluorescence-labeled substrate.     

Sensitive method for the assay of sertindole in plasma by high-performance liquid chromatography and fluorimetric detection

A simple and highly sensitive normal-phase HPLC method is described for determining sertindole concentrations in human plasma using fluorimetric detection. A short C₈ column was used to extract sertindole and the internal standard from plasma; the column was rinsed with acetonitrile, and the analytes were recovered by elution with methanol. This uncommon selectivity between the two solvents allowed clean extraction and near- quantitative recovery of the analytes (> 89%). Separation was done on a 5-μm silica-gel column and detection was performed by fluorimetry, with emission at 340 nm and excitation at 260 nm. The detection and lower quantifiable limits were 0.01 and 0.025 ng/ml, respectively, with no interference from plasma or potential metabolites.     

Automated theophylline assay using gas chromatography and a mass-selective detector

An automated gas chromatographic—mass spectrometric assay for theophylline is described. Theophylline is extracted from plasma or urine (50 μl) and transformed into an N-pentyl derivate. The internal standard used for quantitation is [1,3-¹⁵N, 2-¹³C]theophylline. The detection is performed by monitoring the molecular ions 250 for theophylline and 253 for the internal standard with a quadrupole mass specific detector HP 5790 A. The system has been fully automated: injection, calibration, assay, calculation. The method shows excellent analytical parameters: linearity between 2 and 40 μg/ml; day-to-day reproducibility 1.82% for a concentration of 15 μg/ml; repeatability 0.75% (15 μg/ml) and 0.33% (30 μg/ml). Accuracy is also excellent. Due to the use of an internal standard labelled with stable isotopes, the specificity and high analytical quality of the method make it useful as a reference method to compare with routine theophylline assays.