Determination of free salsolinol concentrations in human urine using gas chromatography—mass spectrometry

The urine concentrations of free salsolinol were determined in six healthy volunteers, using a gas chromatographic—mass spectrometric method with electron-capture negative-ion chemical ionization after derivatization with pentafluoropropionyl anhydride. The sensitivity of this method allows the quantification of salsolinol concentrations of 0.55 pmol/ml. The synthesis of [²H₄]salsolinol from dopamine and [²H₄]acetaldehyde via a Pictet—Spengler condensation is described; [²H₄]salsolinol was used as the internal standard for salsolinol quantification. The urine concentrations of free salsolinol ranged from ca. 1 to 6 pmol/ml.     

High-performance liquid chromatographic determination of acrolein as a marker for cyclophosphamide bioactivation in human liver microsomes

A high-performance liquid chromatographic method for the quantification of acrolein following incubation of cyclophosphamide (CP) with human liver microsomes was developed. Based on the formation of the fluorescent derivative 7-hydroxyquinoline by condensation of acrolein with 3-aminophenol quantitation was performed without prior extraction or other sample cleanup procedures. The method showed sufficient sensitivity with a limit of detection of 5 ng/ml and a limit of quantification of 10 ng/ml. The suitability of the method is shown for enzyme kinetic studies.     

Stereoselective determination of R(−)- and S(+)-MK-571, a leukotriene D4 antagonist,in human plasma by chiral high-performance liquid chromatography

A stereoselective high-performance liquid chromatographic method that utilizes fluorescence detection was developed for the selective and sensitive quantification of R(−)- and S(+)-enantiomers of MK-571 (1), a potent and specific leukotriene D₄ antagonist, in human plasma. Racemic 1 was isolated from the acidified plasma using solid-phase extraction and the resulting residue was successfully reacted with isobutyl chloroformate and R(+)-1-(1-naphthyl)ethylamine in triethylamine—acetonitrile medium to form the diastereomer of each enantiomer. A structural analogue of 1 was used as internal standard. The derivatized sample was dissolved in 1,1,2-trichlorotrifluoroethane and an aliquot was chromatographed on a (R)-urea chiral column using a mobile phase containing 89% triethylamine—pentane (3:1000, v/v), 10% 2-propanol, and 1% acetonitrile at a flow-rate of 1.5 ml/min. The fluorescence response (excitation wavelength, 350 nm; emission wavelength, 410 nm) was linear (r²>0.999) for concentrations of enantiomers of 1 from 0.05 μg/ml, the lowest quantitation limit, up to 2.5 μg/ml. Intra-day coefficients of variation at 0.05 μg/ml were 2.4% for the R(−)-isomer and 2.0% for S(+)-isomer. The corresponding inter-day coefficients of variation for R(−)- and S(+)-1 were 2.6 and 3.6%, respectively. The utilit of the methodology was established by analysis of plasma samples from male volunteers receiving single intravenous and oral doses of racemic 1.     

Quantification of indol-3-yl acetic acid in pea and maize seedlings by gas chromatography-mass spectrometry

A procedure is described for the identification and quantification of IAA in plant tissues by GC/MS analysis of the N-heptafluorobutyryl ethyl ester of IAA using [²H₅]IAA as an internal standard. The detection limit is ca 3 pmol IAA/tissue sample. By using this method, IAA levels of 30–90 pmol/g fr. wt were obtained for dark-grown Pisum sativum epicotyls and 71–199 pmol/g fr. wt for dark-grown Zea mays seedlings. When either methanol or ethanol was used as extraction solvent, some esterification of IAA during sample preparation was observed. No evidence for the natural occurrence of methyl or ethyl esters of IAA in Pisum sativum seedlings was found.     

High-performance liquid chromatographic assay for the determination of the novel etoposide derivative dimethylaminoetoposide (NK611) and its metabolites in urine of cancer patients

A simple, reproducible and specific urine assay for the novel epipodophyllotoxin derivative dimethylaminoetoposide (NK611, I) its picro form (III), the N-demethyl metabolite (II) and its picro form (IV) is reported. The method involves the addition of Pr-NK611 as internal standard, chloroform extraction and HPLC separation on a Nova-Pak C₁₈ column with a mobile phase of acetonitrile-0.05 M KH₂PO₄ (pH 6.4) (23:77, v/v). UV detection was used with absorbance monitored at 205 nm and the limit of quantification was 100 ng/ml. The intra- and inter-day precisions were within the ranges 1.1–3.4% and 1.9–2.4% for all analytes and the accuracy was 101–107%. The extraction recovery was more than 88% for I, II and IV and more than 83% for III. The assay is applicable to the urinary monitoring of I–IV in clinical pharmacokinetic investigations.     

Simultaneous determination of a potassium channel opener and its metabolite in rat plasma with column-switching liquid chromatography using atmospheric pressure chemical ionisation

A specific LC–MS assay was developed for simultaneous determination of Ro 31-7837 (I) and its metabolite Ro 31-6930 (II) in rat plasma, using on-line SPE by column-switching reversed-phase HPLC combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry for detection in the selected reaction monitoring mode. The method involved precipitation of plasma proteins with ethanol and automatic injection of a 1-ml aliquot of the supernatant onto a standard bore trapping column (LC-ABZ, 20×4.6 mm) for compound retention. Using the backflush mode, the analytes were transferred onto the analytical column (Kromasil C₁₈, 125×4.0 mm) for chromatographic separation and mass spectrometric detection. The mean precision and accuracy for I and II in the concentration range 0.25–100 ng/ml were found to be 3.7% and 101%, and 3.5% and 106%, respectively. The data were assessed from QC samples during the validation phase of the assay. The lower limit of quantification for both I and II was 0.25 ng/ml, using a 0.5-ml plasma aliquot. This LC–MS method provided the requisite specificity, sensitivity, accuracy and precision to assess the pharmacokinetics of the compounds in the rat.     

qNMR: An applicable method for the determination of dimethyltryptamine in ayahuasca,a psychoactive plant preparation

Ayahuasca is an Amazonian plant beverage obtained by infusing the pounded stems of Banisteriopsis caapi in combination with the leaves of Psychotria viridis. P. viridis contains the psychedelic indole N,N-dimethyltryptamine (DMT). This association has a wide range of use in religious rituals around the world. In the present work, an easy, fast and non-destructive method by Nuclear Magnetic Resonance of proton (¹H NMR) for quantification of DMT in ayahuasca samples was developed and validated. 2,5-Dimethoxybenzaldehyde (DMBO) was used as internal standard (IS). For this purpose, the area ratios produced by protons of DMT (N(CH₃)₂) at 2.70 ppm, singlet, (6H) and for DMBO (Ar(OCH₃)₂) at 3.80 and 3.89 ppm, doublet, (6H) were used for quantification. The lower limit of quantification (LLOQ) was 12.5 μg/mL and a good intra-assay precision was also obtained (relative standard deviation < 5.1%). The present ¹H NMR method is not time consuming and can be readily applied to monitor this tryptamine in plant preparations. We believe that qNMR can be used for identification and quantification of many plant-based products and metabolites with important advantages, while comparing with other analytical techniques.     

Assay method for the perfluorooctyl bromide (perflubron) in rat blood by gas chromatography–mass spectrometry

This paper describes a GC–MS method (SIM mode) for the analysis of perfluorooctyl bromide (perflubron, I) in rat blood. The chromatographic separation was performed by injection in the split mode using a CP-select 624 CB capillary column. Following destruction of the emulsion by addition of ethanol, the analytical procedure involves a liquid–liquid extraction with 1,1,2-trichlorotrifluoroethane. The bis(F-butyl)ethene (II) was used as internal standard. Observed retention times were 3.22 min for I and 2.32 min for II. Two calibration curves were used; linear detection responses were obtained for concentrations ranging from 0.009 to 0.9 mg/ml and from 0.9 to 13.5 mg/ml. The extraction efficiency averaged 50% for I and 93% for II. Precision ranged from 0.7 to 14%, and accuracy was between 91 and 109%. The limit of quantification was 9 μg/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay method for use in pharmacokinetic studies.     

Fast Screening of Whole Blood Samples and Pharmaceutical Compounds for Enantiorecognition of Free L‐T3, L‐T4, and D‐T4

A fast screening method of whole blood was proposed for enantiorecognition of free L‐T₃ , L‐T₄, and D‐T₄. Stochastic microsensors based on four inulins (IN, IQ, TEX, and HD) immobilized on diamond paste (DP) were used for recognition of free L‐T₃ , L‐T₄, and D‐T₄. For the enantiorecognition of free L‐T₄ and D‐T₄ in whole blood and pharmaceutical samples, the best microsensor was the one based on TEX/DP (wide linear concentration ranges, and low limits of quantification). The best limit of detection for the assay of free L‐T₃ (400 fmol/L) was recorded using the microsensors based on HD/DP, while for the assay of free L‐T4, and D‐T4 the best limit of determination (1 pmol/L) was recorded using the TX/DP‐based microsensor. For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). For the enantiorecognition of free L‐T₃ in whole blood and pharmaceutical samples the best microsensor was the one based on HD/DP (the wider linear concentration range, and the lower limit of quantification – of pmol/L magnitude order). Free L‐T₃ , L‐T₄, and D‐T₄ were recovered with high reliabilities in whole blood samples (recoveries higher than 99.00%, with RSD values lower than 1.00%) and pharmaceutical samples (recoveries higher than 95.00% with RSD values lower than 1.00%). Chirality 27:973–978, 2015. © 2015 Wiley Periodicals, Inc.     

Stable isotope-dilution liquid chromatography/tandem mass spectrometry method for determination of thyroxine in saliva

A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for the determination of thyroxine (T₄) in human saliva has been developed and validated. The saliva was deproteinized with methanol, purified using a Strata-X? cartridge, and subjected to LC/ESI-MS/MS. Quantification was based on selected reaction monitoring, and [¹³C₆]-T₄ was used as the internal standard. This method allowed the reproducible (intra- and inter-assay relative standard deviations, <4.8%) and accurate (analytical recovery, 96.5–99.6%) quantification of the salivary T₄ using a 400 μl sample, and the limit of quantification was 25.0 pg/ml. A preliminary study using the developed method found that there is a diagnosable difference in the salivary T₄ concentration between the euthyroid subjects and the patients with Graves disease.     

Synthesis and application of a “turn on” fluorescent probe for glutathione based on a copper complex of coumarin hydrazide Schiff base derivative

Discrimination and quantification of intracellular biothiols, such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH) under physiological conditions is significant for academic research and disease diagnosis. A new fluorescent probe (complex 1-Cu²⁺) for discriminate detection of GSH was prepared by copper ions coordinate with coumarin carbohydrazide Schiff base derivative 1. In suitable buffer solution (CH₃CN: HEPES = 3:2, v/v) and under appropriate pH condition (pH = 7.2–7.4), the UV–vis spectroscopy experiments showed that compound 1 and copper ion exhibited a 1:1 ratio binding mode and moderate binding ability. Fluorescence quenching of compound 1 was observed when it complexed with Cu²⁺ ions. An obviously fluorescence restoration appeared after addition of GSH to the solution of probe, which also exhibited a highly selectivity relative to cysteine (Cys) and homocysteine (Hcy) in the amino acid competitive experiments. The minimum detection limit was calculated to 0.12 μM by fluorescent method, which was distinctly below the physiological concentration of GSH in live cells. Its biological application to detect the endogenous GSH was further proved by the HepG2 cell fluorescence image test.     

Highly sensitive quantification of serum malonate, a possible marker for de novo lipogenesis, by LC-ESI-MS/MS

We describe a new sensitive and specific method for the quantification of serum malonate (malonic acid, MA), which could be a new biomarker for de novo lipogenesis (fatty acid synthesis). This method is based upon a stable isotope-dilution technique using LC-MS/MS. MA from 50 μl of serum was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantified by LC-MS/MS using the positive electrospray ionization mode. The detection limit of the DMP-MA was approximately 4.8 fmol (500 fg) (signal-to-noise ratio = 10), which was more than 100 times more sensitive compared with that of MA by LC-MS/MS using the negative electrospray ionization mode. The relative standard deviations between sample preparations and measurements made using the present method were 4.4% and 3.2%, respectively, by one-way ANOVA. Recovery experiments were performed using 50 μl aliquots of normal human serum spiked with 9.6 pmol (1 ng) to 28.8 pmol (3 ng) of MA and were validated by orthogonal regression analysis. The results showed that the estimated amount within a 95% confidence limit was 14.1 ± 1.1 pmol, which was in complete agreement with the observed X¯₀ = 15.0 ± 0.6 pmol, with a mean recovery of 96.0%. This method provides reliable and reproducible results for the quantification of MA in human serum.     

Direct gas chromatographic determination of dechloroethylcyclophosphamide following microsomal incubation of cyclophosphamide

A method for the sensitive determination of dechloroethylcyclophosphamide (3-DCl) in microsomal incubation mixtures was developed. 3-DCl, a side-chain oxidation product of cyclophosphamide (CP), was isolated by extraction with acetic acid ethyl ester following solid-phase extraction on C₈ cartridges. Quantification of the metabolite was performed by direct capillary gas chromatography with a nitrogen-phosphorus detector without prior derivatization. The method showed good sensitivity and reproducibility with a detection limit of 1 ng/ml and a limit of quantification of 5 ng/ml. The suitability of the method is shown for the quantification of 3-DCl following incubation of CP with human liver microsomes.     

Differential pulse polarographic determination of plasma menadione

A differential pulse polarographic assay for plasma vitamin K₃ (menadione) has been developed. Details of the assay are (i) lipid-soluble material is extracted from plasma into ether by the method of Bjornsson et al. [(1978) Thromb. Haemostas.2, 466–473]; (ii) ether is evaporated under nitrogen and the residue is dissolved in the supporting electrolyte, methanol: 0.2 m borate buffer (9:1), pH 6.8; (iii) current height is measured at ?0.32 V vs SCE on the differential pulse polarogram. The lower sensitivity limit of this technique after addition of standard vitamin K₃ to plasma is 0.3 μm; the calibration curve is linear from 0.6 through 10 μm. Two patients treated with a single dose of menadiol sodium diphosphate, 20 mg/M² i.m., achieved measurable plasma vitamin K₃ levels at 0.5 to 1.0 h ranging between 0.5 (0.08 μg/ml) and 2 μm (0.3 μg/ml).     

Application of selected ion monitoring technique for quantitative determination of picomole levels of pyrrolidine in the brain

A specific and sensitive method for analysis of brain pyrrolidine, a volatile amine with potent synaptotropic actions on the peripheral and central nervous systems, was devised. The method involves the isolation of volatile amines by steam distillation and the qualification and quantification of pyrrolidine by gas chromatography/mass spectrometry ($\text{gc}\text{ms}$) including a selected ion monitoring technique with deuterium-labeled pyrrolidine as an internal standard. The lower limit of quantification for the method was 2 pmol, and the mean concentration of pyrrolidine in the rat whole brain was determined to be 95 pmol/g of tissue.     

Xanthine oxidase inhibitors from an endophytic fungus Lasiodiplodia pseudotheobromae

Two new compounds, lasdiplactone (1) and lasdiploic acid (2) and one known compound 3 were isolated from the chloroform extract of cell free filtrate of the endophytic fungus Lasiosdiplodia pseudotheobromae. The structures of new compounds were determined by interplay of spectral techniques (IR, mass, ¹H NMR, ¹³C NMR, DEPT, and 2D NMR). The absolute configuration at C-4 position of 1 was established as S using a process similar to modified Mosher’s method. The absolute configuration of 2 was established by comparing its ECD spectrum with the calculated ECD spectra of all possible isomers. In the in vitro XO inhibition assay, the highest inhibition was exhibited by 3 with an IC₅₀ of 0.38 ± 0.13 μg/ml, followed by 2 with an IC₅₀ of 0.41 ± 0.1 μg/ml and the least in 1. The oxidized form of 1 also showed high XO inhibition with IC₅₀ of 0.35 ± 0.13 μg/ml.     

Validation of a high-performance liquid chromatographic assay for the quantification of adenovirus type 5 particles

An anion-exchange–high-performance liquid chromatography (AE–HPLC) method for the quantification of adenovirus type 5 (Ad5) total particles was validated according to performance criteria of precision, specificity, linearity of calibration and range, limit of detection, limit of quantification, accuracy and recovery. The viral particles were detected by absorbance at 260 nm using photodiode array detector (PDA). Cesium chloride (CsCl) purified Ad5 and lysate samples were used for the validation of the method. Relative standard deviations (RSDs) for the inter-day, intra-day precision and reproducibility for both the lysate and the Ad5 standard were less than 10 and 2% for the peak area and retention time, respectively. The method was specific for Ad5 which was eluted at 8.0 min. The presence of DNA does not affect the recovery of Ad5 particles for accurate quantification. Based on the error in prediction to be less than 10%, the working range was established between 2×10¹⁰ and 7×10¹¹ VP/ml with correlation coefficient of 0.99975, standard deviation of 6.14×10⁹ VP/ml and a slope of 3.04×10⁵ VP/ml. The recovery of the method varied between 88 and 106% in all of the lysate samples investigated which is statistically similar to 100% recovery at 95% confidence interval.     

Quantum dot-based assay for Cu quantification in bacterial cell culture

A simple and sensitive method for quantification of nanomolar copper with a detection limit of 1.2 × 10⁻¹⁰ M and a linear range from 10⁻⁹ to 10⁻⁸ M is reported. For the most useful analytical concentration of quantum dots, 1160 μg/ml, a 1/K_sv value of 11 μM Cu²⁺ was determined. The method is based on the interaction of Cu²⁺ with glutathione-capped CdTe quantum dots (CdTe–GSH QDs) synthesized by a simple and economic biomimetic method. Green CdTe–GSH QDs displayed the best performance in copper quantification when QDs of different sizes/colors were tested. Cu²⁺ quantification is highly selective given that no significant interference of QDs with 19 ions was observed. No significant effects on Cu²⁺ quantification were determined when different reaction matrices such as distilled water, tap water, and different bacterial growth media were tested. The method was used to determine copper uptake kinetics on Escherichia coli cultures. QD-based quantification of copper on bacterial supernatants was compared with atomic absorption spectroscopy as a means of confirming the accuracy of the reported method. The mechanism of Cu²⁺-mediated QD fluorescence quenching was associated with nanoparticle decomposition.     

Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.     

Determination of pyridinium crosslinks in plasma and serum by high-performance liquid chromatography

A chromatographic method for the determination of pyridinoline (Pyr) and deoxypyridinoline (Dpyr) in serum and plasma is described. The analytical procedure involved plasma or serum purification by ultrafiltration (20 000 relative molecular mass cut-off) under centrifugation at 2500 g for 4 h, as an innovative step. Analysis was done by isocratic high-performance liquid chromatography with fluorescence detection. The linearity of the method was tested from 0.6 to 15 pmol/ml and 0.12 to 3 pmol/ml for Pyr and Dpyr, respectively. The detection limit was 60 fmol/ml for both crosslinks. Except for Dpyr in plasma (coefficient of variation 19.9%), intra-assay variation was always below 10% in serum and plasma. The method has been applied to the quantification of crosslinks in serum and plasma of healthy volunteers and also in mouse and rat plasma. Serum proved to be the most suitable biological fluid for the systemic measurement of these compounds in humans and under the experimental conditions used, contained an average of 3.62 ± 0.65 and 0.7 ± 0.18 pmol/ml Pyr and Dpyr, respectively.