Mass spectrometry‐based absolute quantification of microsomal cytochrome P450 2D6 in human liver

We describe the assay of the human cytochrome P450 2D6 in a set of 30 genotyped liver samples using the ‘absolute quantification’ (AQUA) technique. We found approximately 30 fmol CYP2D6 per μg of microsomal protein, with the values spanning from 0 to nearly 80 fmol/μg. This is greater by a factor of 5–10 from the compared to the currently accepted value, which was around 5 fmol/μg. Our results thus suggest that the amount of cytochrome P450 (CYP) enzymes in liver have to be reassessed. We used quantitative Western blotting, calibration standards and activity assays, to validate the results. Our results show, that using the AQUA technique a true assay of CYP2D6 in human liver was possible.     

Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors

We have obtained, for the first time, a quantitative protein expression profile of membrane transporters and receptors in human brain microvessels, that is, the blood-brain barrier (BBB). Brain microvessels were isolated from brain cortexes of seven males (16-77 years old) and protein expression of 114 membrane proteins was determined by means of a liquid chromatography-tandem mass spectrometric quantification method using recently established in-silico peptide selection criteria. Among drug transporters, breast cancer resistance protein showed the most abundant protein expression (8.14 fmol/μg protein), and its expression level was 1.85-fold greater in humans than in mice. By contrast, the expression level of P-glycoprotein in humans (6.06 fmol/μg protein) was 2.33-fold smaller than that of mdr1a in mice. The organic anion transporters reported in rodent BBB, that is, multidrug resistance-associated protein, organic anion transporter and organic anion-transporting polypeptide family members, were under limit of quantification in humans, except multidrug resistance-associated protein 4 (0.195 fmol/μg protein). Among detected transporters and receptors for endogenous substances, the glucose transporter 1 level was similar to that of mouse, while the L-type amino acid transporter 1 level was fivefold smaller than that of mouse. These findings should be useful for understanding human BBB function and its differences from that in mouse.     

Simultaneous quantification of components of neoglycolipid-coated liposomes using high-performance liquid chromatography with evaporative light scattering detection

Liposomes composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol and a neoglycolipid, mannopentaose-conjugated dipalmitoylphosphatidylethanolamine (Man5-DPPE), have been shown to have a strong adjuvant effect in inducing the antigen-specific cellular immunity. In this study, a rapid and simple analytical method using a HPLC system with an evaporative light scattering detector was developed for simultaneous quantification of the liposome components Man5-DPPE, cholesterol and DPPC. The chromatographic separation of these components was performed using a trimethylsilane column with an isocratic mobile phase of chloroform–methanol–water (1:33:6, v/v) after disrupting the liposomes with chloroform–methanol–water (10:10:3, v/v). This HPLC method provided sufficient reproducibility and linearity of calibration curves for the quantification of the liposome constituents. In addition, this method can be used for the quantification of various neoglycolipids with different carbohydrate structures.     

Simple Isocratic HPLC Method for Determination of Enantiomeric Impurity in Besifloxacin Hydrochloride

Besifloxacin is a unique chiral broad‐spectrum flouroquinolone used in the treatment of bacterial conjunctivitis. R‐form of besifloxacin hydrochloride shows higher antibacterial activity as compared to the S‐isomer. Therefore, it is necessary to establish chiral purity. To establish chiral purity a high‐performance liquid chromatography (HPLC) method for determination of R‐besifloxacin and S‐besifloxacin (BES impurity A) was developed and validated for in‐process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD), and lower limit of quantification (LOQ) were determined according to International Council for Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on Chiralpak AD‐H (250 x 4.6 mm, 5 μm) column using n‐heptane: ethanol: ethylenediamine: acetic acid (800:200:0.5:0.5) (v/v/v/v) as the mobile phase in an isocratic elution. The eluents were monitored by UV/Visible detector at 290 nm. The resolution between S‐isomer and besifloxacin hydrochloride was more than 2.0. Based on a signal‐to‐noise ratio of 3 and 10 the LOD of besifloxacin was 0.30 μg/mL, while the LOQ was 0.90 μg/mL. The calibration curves were linear in the range of 0.9–7.5 μg/mL. Precision of the method was established within the acceptable range. The method was suitable for the quality control enantiomeric impurity in besifloxacin hydrochloride. Chirality 28:628–632, 2016. © 2016 Wiley Periodicals, Inc.     

Development of an analytical method for the determination of sterigmatocystin in grains using LCMS after immunoaffinity column purification

The mycotoxin sterigmatocystin (STC) is produced mainly by some Aspergillus and Penicillium fungi; it naturally contaminates cereals, peanuts, and products derived from these crops, and is both mutagenic and carcinogenic. As an intermediate of aflatoxin (AF) biosynthesis, its structure is similar to that of AF. Although immunoaffinity columns (IACs) are a popular approach to sample clean-up, no IAC is commercially available for STC, but a commercially available IAC for AF shows cross reactivity to STC. We here developed a new method for analyzing STC in grains using such an IAC and liquid chromatography mass spectrometry (LCMS), and validated this method using six different grains. The STC limit of detection (signal-to-noise ratio, S/N?=?3) was 2.5 pg (1.0 μg/kg in the product), and the calibration curve was linear in the range of 7.5–375 pg (3.0–150 μg/kg in the product). The within-day recovery of STC from samples spiked with STC at 5.0 and 50 μg/kg was 83.2–102.5 % and the RSDr (relative standard deviation of repeatability) of these samples was 1.9–6.5 %; the RSDr of STC-pretreated grain samples was 3.1–14.0 %. Average recovery of STC from samples spiked with STC in the range of 5.0–100 μg/kg STC was 83.2–102.5 %, with an RSDr of 0.24–6.5 %; the RSDr of STC-pretreated grain samples was 2.4–14.0 %. In an intermediate precision study, the average STC recovery from STC-spiked samples by three analysts was 95.2–107.5 %, with RSDRi (intermediate precision) of 4.0–7.1 %; the RSDRi of the STC-pretreated samples was 4.8–10.4 %. Thus, the proposed method was effective for STC analysis in grains, and holds potential for a novel application of a commercial IAC, intended for AFs, in STC analysis.     

Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for the sensitive determination of folates in rice

High-performance liquid chromatography, coupled to tandem mass spectrometry (HPLC–MS/MS) has been established as the method of choice for the sensitive and simultaneous determination of different folates in a particular matrix, especially when only minute quantities of material are available. Using a previously developed and validated HPLC–MS/MS method as a starting point, we here report on the development and validation of an ultra-performance liquid chromatography (UPLC–MS/MS) method for analysis of folates in rice, which allows higher throughput and better resolution. UPLC was performed under gradient conditions on an Acquity HSS T3 column, followed by tandem mass spectrometry detection. The method was validated based on linearity, sensitivity, precision, accuracy and matrix effects. The limits of detection and the lower limits of quantification varied between 0.06 and 0.45 μg/100 g and 0.12 and 0.91 μg/100 g, respectively. Two linear calibration curves were established, one for the low and the other for the high concentration range. Analysis of the distribution and levels of folates in wild-type and folate-biofortified rice showed up to 50-fold enrichment in biofortified rice, with total folate levels of up to 900 μg/100 g rice. This is the first successful implementation of a UPLC method for the rapid and sensitive quantitative determination of folates in plant material.     

Micro-RNA quantification using DNA polymerase and pyrophosphate quantification

A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20–100 μg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/μg RNA and 0.71 fmol/μg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays.     

Determination of biogenic amines in infusions of tea (Camellia sinensis) by HPLC after derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl)

The reagent 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) was used for the pre-column derivatization of the biogenic amines (BAs) cadaverine (Cad), histamine (Him), octopamine (Ocp), phenylethylamine (Pea), putrescine (Put), spermidine (Spd), spermine (Spm), tyramine (Tym) and the internal standard 1,6-diaminohexane (Dhx). The resulting Fmoc-derivatives were resolved by high-performance liquid chromatography on a Superspher^? C₁₈ column using a binary gradient generated from sodium acetate and acetonitrile. For quantification, the fluorescence of derivatives was used at 263?nm excitation and 313?nm emission wavelength. This approach was applied to free BAs extractable with boiling water from 14 black, 5 green, 1 Oolong, and 1 instant tea. Infusions were prepared by adding 35?ml boiling water to one gram of tea and extracted for 20?min. In the Oolong tea and two black teas, no BAs could be detected. Limits of detection were 0.07–1.0?pmol for BAs at signal-to-noise ratio 3:1. Besides most abundant Tym and Spm lower quantities of Pea, Put, and Spd were detected, albeit not in all teas. Quantities of Tym ranged from 16 to 431?μg Tym/L infusion (1.1–25.3?μgTym/g tea) and 31 to 319?μg Spm/L infusion (1.5–16.9?μg Spm/g tea). In none of the teas, Him was detected. Owing to the low amounts of free BAs in tea infusions, no health risks are to be expected even on consumption of large quantities of tea as beverage.     

Liquid chromatography tandem mass spectrometry method for the quantitation of NTBC (2-(nitro-4-trifluoromethylbenzoyl)1,3-cyclohexanedione) in plasma of tyrosinemia type 1 patients

In this study, we describe a bioanalytical method for quantification of NTBC in plasma of patients with hereditary tyrosinemia type 1 (HT-1) using high-performance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). After protein precipitation with acetonitrile including Mesotrione as internal standard, separation of NTBC was achieved by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in selected reaction monitoring (SRM) mode. NTBC recovery in the developed method was found to be more than 90%. The lower limit of quantification was calculated to be 0.35 μM. The intra-day and inter-day precision of three different quality control samples (measured as RSD%) was less than 10% and 15%, respectively. The standard calibration curves showed good linearity within the range of 2.5–40 μM and the determined correlation coefficients were r² ≥ 0.995. This presented method is rapid, sensitive, specific and suitable for clinical practice and research.     

The determination of allopurinol and oxipurinol in human plasma and urine

A method is described for allopurinol and oxipurinol assay within human plasma and urine in the range expected during therapy. The method is based on high-performance ion-exchange chromatography following an efficient sample purification step using Chelex-100 resin in the Cu²⁺ form. Linear calibration curves are produced for allopurinol over the range 0.05–10 μmole/1 (0.068–1.36 μg/ml) in plasma and 0.005–1 mmole/1 (0.68–136 μg/ml) in urine and for oxipurinol 0.5–100 μmole/1 (0.076–15.2μg/ml) in plasma and 0.1–2 mmole/1 (15.2–304 μg/ml) in urine.     

Proteome analysis of rat serum proteins adsorbed onto synthetic octacalcium phosphate crystals

The present study was designed to determine which proteins are selectively adsorbed onto two bone substitute materials, octacalcium phosphate (OCP) and hydroxyapatite (HA) crystals, from rat serum by proteome analysis. Ground crystals of synthetic OCP and commercially available sintered HA, with the same surface area, were incubated in rat serum proteins at 37 °C for 24 h. The proteins from the crystals extracted with guanidine–HCl–EDTA were listed on the basis of the results of liquid chromatography tandem mass spectrometry (LC/MS/MS). A total of 138 proteins were detected from OCP; 103 proteins were detected from HA. Forty-eight proteins were from both crystals. A quantitative analysis of the proteins detected was performed for the extracted two bone formation-related proteins apolipoprotein E (Apo E), a protein known to promote osteoblast differentiation, and complement 3 (C3). HA adsorbed C3 (3.98 ± 0.03 fmol/μg protein) more than OCP (1.81 ± 0.07 fmol/μg protein) did, while OCP adsorbed Apo E (2.42 ± 0.03 fmol/μg protein) more than HA (1.21 ± 0.01 fmol/μg protein) did even after deleting the high-abundance proteins, such as albumin. The results demonstrated that OCP exhibits a similar property but distinct capacity with HA in adsorbing bone formation-related proteins from the serum constituents.     

Determination of anandamide and other fatty acyl ethanolamides in human serum by electrospray tandem mass spectrometry

We developed a new selective liquid chromatography-electrospray ionization-tandem mass spectrometry method for the identification and quantification of anandamide (AEA), an endogenous cannabinoid receptor ligand, and other bioactive fatty acid ethanolamides (FAEs) in biological samples. Detection limit (0.025 pmol for AEA and 0.1 pmol for palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)) and quantification limit (0.2 pmol for AEA and 0.4 pmol for OEA and PEA) were in the high fmol to low pmol range for all analytes. Linear correlations (r(2)=0.99) were observed in the calibration curves for standard AEA over the range of 0.025-25 pmol and for standard PEA and OEA over the range of 0.1-500 pmol. This method provides a time-saving and sensitive alternative to existing methods for the analysis of FAEs in biological samples.     

Determination of free and liposomal Amphotericin B in human plasma by liquid chromatography–mass spectroscopy with solid phase extraction and protein precipitation techniques

Amphotericin B is available in various drug delivery systems such as cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The separation and measurement of free drug (drug which is not bound with liposomal lipids) and liposomal drug (drug which is entrapped in liposomes) in the human plasma after injection of liposomal Amphotericin B is of prime importance due to toxicity concerns. A robust, specific and sensitive method has been developed to effectively separate and then quantify the free drug and liposomal drug, present in human plasma. This method utilizes solid phase extraction Oasis HLB cartridges, which retains the free drug and the liposomal Amphotericin B was eluted from the cartridge in first step. The eluted liposomal Amphotericin B was then extracted from lipids by protein precipitation method using 2% dimethylsulfoxide (DMSO) in acetonitrile. After separation and extraction, the quantification of free and liposomal fractions of Amphotericin B was performed by HPLC–MS–MS technique. The chromatographic separation was performed using Chromolith Performance RP 18e column. The mobile phase composed of 5 mM ammonium acetate, methanol and acetonitrile and a gradient elution program was used. The calibration curves were found to be linear for free Amphotericin B (0.25–15.0 μg/ml) and liposomal Amphotericin B (1.0–100.0 μg/ml). The recovery was about 96% for free Amphotericin B and about 92% for liposomal Amphotericin B. Recoveries were consistent over the linearity ranges defined. The intra-batch and inter-batch accuracy and precision fulfilled the international requirements. The stability of free and liposomal Amphotericin B was assessed under different storage conditions.     

A simple glutathione transferase-based colorimetric endpoint assay for insecticide detection

The natural ability of the detoxification enzymes glutathione transferases (GSTs) to interact with xenobiotics can be used for the production of colorimetric assays. Detection is usually based on the inhibition of the GST-catalysed reaction, with detection achieved spectrophotometrically or electrochemically. Here we have adopted a chromogenic (visual) activity assay for screening GSTs with alkyltransferase activity for iodoalkene substrates for detection of insecticides. We screened a number of GSTs from insecticide resistant mosquito species for their ability to catalyse iodoalkane biotransformation reactions. AaGSTE2 was found to metabolise iodoethane with high turnover, which resulted in a dark blue colour in the enzymatic reaction. Following assay optimisation we exploited the high recognition affinity of the AgGSTE2 for insecticides to develop a novel colorimetric detection assay for organochlorine and pyrethroid quantification. Calibration curves were obtained for permethirn, deltamethrin, λ-cyhalothrin and DDT, with useful concentration ranges of 0–40 μg/ml (0–100 μM), 0–50 μg/ml (0–100 μM), 0–100 μg/ml (0–220 μM), and 0–50 μg/ml (0–140 μM), respectively. The assay was validated with extracts from insecticide sprayed surfaces and found to be reproducible and reliable compared with HPLC. The assay is therefore suitable for monitoring insecticide residues in insecticide treated materials, and therefore has potential for insect vector control operations.     

Spectrofluorimetric determination of eptifibatide in human plasma and dosage form

A spectrofluorimetric method for the determination of eptifibatide is presented based on its native fluorescence. The type of solvent and the wavelength of maximum excitation and emission were carefully selected to optimize the experimental conditions. Under the specified experimental conditions, the linearities obtained between the emission intensity and the corresponding concentrations of eptifibatide were in the range 0.1–2.5 μg/ml for the calibration curve constructed for direct determination of eptifibatide in dosage form and 0.05–2.2 μg/ml for the calibration curve constructed in spiked human plasma with a good correlation coefficient (r > 0.99). The lower limit of quantification for the calibration curve constructed in human plasma was 0.05 μg/ml. Recovery results for eptifibatide in spiked plasma samples and in dosage form, represented as mean ± % RSD, were 95.17 ± 1.94 and 100.29 ± 1.33 respectively. The suggested procedures were validated according to the International Conference on Harmonization (ICH) guidelines for the direct determination of eptifibatide in its pure form and dosage form and United States Food and Drug Administration (US FDA) Guidance for Industry, Bioanalytical Method Validation for the assay of eptifibatide in human plasma.     

Quantification of strontium in human serum by ICP-MS using alternate analyte-free matrix and its application to a pilot bioequivalence study of two strontium ranelate oral formulations in healthy Chinese subjects

A rapid, sensitive and accurate ICP-MS method using alternate analyte-free matrix for calibration standards preparation and a rapid direct dilution procedure for sample preparation was developed and validated for the quantification of exogenous strontium (Sr) from the drug in human serum. Serum was prepared by direct dilution (1:29, v/v) in an acidic solution consisting of nitric acid (0.1%) and germanium (Ge) added as internal standard (IS), to obtain simple and high-throughput preparation procedure with minimized matrix effect, and good repeatability. ICP-MS analysis was performed using collision cell technology (CCT) mode. Alternate matrix method by using distilled water as an alternate analyte-free matrix for the preparation of calibration standards (CS) was used to avoid the influence of endogenous Sr in serum on the quantification. The method was validated in terms of selectivity, carry-over, matrix effects, lower limit of quantification (LLOQ), linearity, precision and accuracy, and stability. Instrumental linearity was verified in the range of 1.00–500 ng/mL, corresponding to a concentration range of 0.0300–15.0 μg/mL in 50 μL sample of serum matrix and alternate matrix. Intra- and inter-day precision as relative standard deviation (RSD) were less than 8.0% and accuracy as relative error (RE) was within ±3.0%. The method allowed a high sample throughput, and was sensitive and accurate enough for a pilot bioequivalence study in healthy male Chinese subjects following single oral administration of two strontium ranelate formulations containing 2 g strontium ranelate.     

Development of a multipurpose time-resolved fluorescence immunoassay for rat insulin

We present a time-resolved fluorescence immunoassay (TR-FIA) for the measurement of rat insulin in cell extracts and culture media. This assay is based on the binding of two monoclonal antibodies to different parts of the insulin molecule in a 96-well microtiter plate. For the detection, europium-labeled streptavidin that interacts with the second biotinylated antibody is used. Samples of 25 μl could be analyzed in less than 2 days with a measuring range between 5 and 1250 pg (0.2-50 μg/L or 34.4-8600 pM). The inter- and intraassay percentage coefficients of variation were less than 8.3 and 5.1, respectively. Recoveries of 0.48 to 40 μg/L rat insulin, added to culture medium, ranged between 94 and 107%. Results were significantly correlated with those of an in-house radioimmunoassay (RIA) for rodent insulin (P < 0.0001, r² = 0.99). The TR-FIA method had a similar detection limit (0.16 μg/L), but its working range was at least 5-fold larger. Additional advantages include the lower cost, the applicability to measurements in tissue and serum, and the quantification of insulin from other species.     

Characterization of a rapid and reliable method for iodide biomonitoring in serum and urine based on ion chromatography–ICP-mass spectrometry

An appropriate and controlled supply of thyroid hormones is vital for proper body function. In turn, an appropriate synthesis of T3 and T4 in the thyroid gland is dependent on a sufficient and balanced iodide concentration in blood serum. Due to widespread iodine deficiency or some cases of iodine over exposure, iodide biomonitoring in serum is important and it is that biomonitoring approach being closest to the bioavailable I⁻ supply for the thyroid gland. Therefore, this paper describes a biomonitoring method for iodide determination in serum based on ion chromatography–inductively coupled plasma mass spectrometry (IC–ICP-MS). Since in literature only very few data are available on iodide in serum but many in urine the method is also extended to I⁻ monitoring in urine. The method was additionally designed to have short analysis time (8 min) for increased sample throughput, good precision in serial measurement (serum: 4.86%; urine: 1.4%), and day-to-day determination (serum: 5.7%; urine: 2.28%), high accuracy (serum: 105%; urine: 101%) and good recovery (serum: 102%; urine: 99%) even in matrix-rich samples at low I⁻ concentration. Also, investigations were performed to elucidate whether internal standardization during chromatography, sample preparation for protein-matrix removal or matrix-matched calibration are advantageous for analytical performance. Finally, limits of detection (3σ) of 0.12 μg/L or 0.05 μg/L (serum or urine) and limit of quantification (10σ) of 0.39 μg/L or 0.17 μg/L (serum or urine) were achieved.     

An easy,inexpensive, and sensitive method for the quantification of chitin in insect peritrophic membrane by image processing

ABSTRACT

Chitin, poly (β-(1→4)-N-acetyl-d-glucosamine), is an important biopolymer for insects that is utilized as a major component of peritrophic membrane. The chitin content in peritrophic membrane is of expedient interest from a pest control perspective, although it is hard to quantify chitin. In this study, we establish a facile method for the quantification of chitin in peritrophic membrane by image processing. In this method, chitin was indirectly quantified using chitosan–I₃^? complex, which exhibited a specific red-purple color. A calibration curve using a chitosan solution showed good linearity in a concentration range of 0.05–0.5 μg/μL. We quantified the amount of chitin in peritrophic membrane of Spodoptera litura (Lepidoptera: Noctuidae) larvae using this method. Throughout the study, only common inexpensive regents and easily attainable apparatuses were employed. This method can be easily applied to the sensitive quantification of the amounts of chitin and chitosan in materials by wide range of researchers.

Abbreviations: LOD: limit of detection; LOQ: limit of quantification; ROI: region of interest; RSD: relative standard deviation.     

Evaluation of Calcium and Magnesium in Scalp Hair Samples of Population Consuming Different Drinking Water: Risk of Kidney Stone

The objective of this study was to examine the relationship between calcium (Ca) and magnesium (Mg) in underground water (UGW), bottled mineral water (BMW), and domestic treated water (DTW) with related to risk of kidney stones. The water samples were collected from different areas of Sindh, Pakistan. The scalp hair samples of both genders, age ranged 30–60 years, consuming different types of water, have or have not kidney disorders, were selected. The Ca and Mg concentrations were determined in scalp hair of study subjects and water by flame atomic absorption spectroscopy. The Ca and Mg contents in different types of drinking water, UGW, DTW, and BMW, were found in the range of 79.1–466, 23.7–140, and 45–270 mg/L and 4.43–125, 5.23–39.6, and 7.16–51.3 mg/L, respectively. It was observed that Ca concentration in the scalp hair samples of kidney stone patients consuming different types of drinking water was found to be higher (2,895–4721 μg/g) while Mg level (84.3–101 μg/g) was lower as compare to referents subjects (2,490–2,730 μg/g for Ca, 107–128 μg/g for Mg) in both genders. The positive correlation was found between Ca and Mg levels in water with related to kidney stone formations in population, especially who consumed underground water. A relative risk and odd ratio were calculated; the relative risk had a strong positive association with incidence of kidney stone which depends on types of drinking water.