Detection of HbD Punjab Trait in Saurashtra Region of Gujarat by HPLC Method.

The HbD Punjab trait is a less common hemoglobin variant in the Saurashtra region of Gujarat. This patient presented to the General Medicine and Biochemistry Department at All India Institute of Medical Sciences, Rajkot, Hospital, for the HbA1c analysis by high-performance liquid chromatography (HPLC). The patient was 52 years old, a known case of type 2 diabetes mellitus and hypertension, and presented with complaints of generalized malaise and fatigability for the previous ten to twelve months. On physical examination, there was no evidence of pallor, lymphadenopathy, splenomegaly, or hepatomegaly. Peripheral blood smear revealed normocytic normochromic RBC, and platelets were adequate and normal in morphology. The HPLC analysis revealed heterozygous for hemoglobin D (HbD Punjab trait), and false high HbA1c corresponds to fasting plasma glucose (FPG) levels due to abnormal separation. However, genetic counseling is highly recommended to detect any potential reproductive risk factors.

2,5-Dimethoxy-4-iodoamphetamine and altanserin induce region-specific shifts in dopamine and serotonin metabolization pathways in the rat brain.

PURPOSE: For understanding the neurochemical mechanism of neuropsychiatric conditions associated with cognitive deficits it is of major relevance to elucidate the influence of serotonin (5-HT) agonists and antagonists on memory function as well dopamine (DA) and 5-HT release and metabolism. In the present study, we assessed the effects of the 5-HT2A receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) and the 5-HT2A receptor altanserin (ALT) on object and place recognition memory and cerebral neurotransmitters and metabolites in the rat. METHODS: Rats underwent a 5-min exploration trial in an open field with two identical objects. After systemic injection of a single dose of either DOI (0.1 mg/kg), ALT (1 mg/kg) or the respectice vehicle (0.9 % NaCl, 50 % DMSO), rats underwent a 5-min test trial with one of the objects replaced by a novel one and the other object transferred to a novel place. Upon the assessment of object exploration and motor/exploratory behaviors, rats were sacrificed. DA, 5-HT and metabolite levels were analyzed in cingulate (CING), caudateputamen (CP), nucleus accumbens (NAC), thalamus (THAL), dorsal (dHIPP) and ventral hippocampus (vHIPP), brainstem and cerebellum with high performance liquid chromatography. RESULTS: DOI decreased rearing but increased head-shoulder motility relative to vehicle. Memory for object and place after both DOI and ALT was not different from vehicle. Network analyses indicated that DOI inhibited DA metabolization in CING, CP, NAC, and THAL, but facilitated it in dHIPP. Likewise, DOI inhibited 5-HT metabolization in CING, NAC, and THAL. ALT facilitated DA metabolization in the CING, NAC, dHIPP, vHIPP, and CER, but inhibited it in the THAL. Additionally, ALT facilitated 5-HT metabolization in NAC and dHIPP. CONCLUSIONS: DOI and ALT differentially altered the quantitative relations between the neurotransmitter/metabolite levels in the individual brain regions, by inducing region-specific shifts in the metabolization pathways. Findings are relevant for understanding the neurochemistry underlying DAergic and/or 5-HTergic dysfunction in neurological and psychiatric conditions.

A Cost-Effective and Sensitive Method for the Determination of Lincomycin in Foods of Animal Origin Using High-Performance Liquid Chromatography.

BACKGROUND: Lincomycin (LIN) is extensively used for treating diseases in livestock and promoting growth in food animal farming, and it is frequently found in both the environment and in food products. Currently, most of the methods for detecting lincomycin either lack sensitivity and precision or require the use of costly equipment such as mass spectrometers. RESULT: In this study, we developed a reliable high-performance liquid chromatography-ultraviolet detection (HPLC-UVD) method and used it to detect LIN residue in 11 types of matrices (pig liver and muscle; chicken kidney and liver; cow fat, liver and milk; goat muscle, liver and milk; and eggs) for the first time. The tissue homogenates and liquid samples were extracted via liquid-liquid extraction, and subsequently purified and enriched via sorbent and solid phase extraction (SPE). After nitrogen drying, the products were derivatized with p-toluene sulfonyl isocyanic acid (PTSI) (100 µL) for 30 min at room temperature. Finally, the derivatized products were analyzed by HPLC at 227 nm. Under the optimized conditions, the method displayed impressive performance and demonstrated its reliability and practicability, with a limit of detection (LOD) and quantification (LOQ) of LIN in each matrix of 25-40 µg/kg and 40-60 µg/kg, respectively. The recovery ranged from 71.11% to 98.30%. CONCLUSIONS: The results showed that this method had great selectivity, high sensitivity, satisfactory recovery and cost-effectiveness-fulfilling the criteria in drug residue and actual detection requirements-and proved to have broad applicability in the field of detecting LIN in animal-derived foods.

Pharmacokinetic profile of phenytoin in dried blood spot with high-performance liquid chromatography-photodiode array.

Phenytoin is a first-line antiepileptic drug with narrow therapeutic range and follows non-linear pharmacokinetics. Pharmacokinetics of phenytoin have been studied in plasma matrix before, however, there were several disadvantages. This study aimed to obtain partial validation data of the analytical method and the pharmacokinetic profile of phenytoin in Dried Blood Spot (DBS) of six healthy subjects. DBS has the advantage of only requiring small sample volumes and could be transported more efficiently. Phenytoin along with carbamazepine as the chosen internal standard was analyzed with a reversed-phase high performance-liquid chromatography system and a photodiode array detector at 205 nm. The results of partial validation, which evaluated the linearity, within-run accuracy, and precision, were within the criteria acceptance range. The pharmacokinetic profile showed that average AUC0-t was 83.81 ± 37.32 µg.h/mL and AUC0-∞ was 83.65 ± 38.89 µg.h/mL with an average ratio of 93%. Previous study quantifying phenytoin in the plasma matrix found the average AUC0-t was 39.41 ± 8.57 µg.h/mL and AUC0-∞ was 42.94 ± 9.55 µg.h/mL. Despite the difference between parameters of phenytoin analyzed in DBS and plasma matrices, the pharmacokinetic profiles obtained from both matrices were similar indicated by comparable concentration-time curves, thus, proving that DBS matrix can be used interchangeably with the plasma matrix as a more comfortable and effective alternative to phenytoin quantification in blood.

Discovery of Unprecedented Human Stercobilin Conjugates.

Two unique metabolites (M18 & M19) were detected in feces of human volunteers dosed orally with [14C]inavolisib with a molecular ion of parent plus 304 Da. They were generated in vitro by incubation with fecal homogenates and we have evidence that they are formed chemically and possibly enzymatically. Structural elucidation by high resolution mass spectrometry and NMR spectroscopy showed that the imidazole ring of inavolisib was covalently bound to partial structures derived from stercobilin, an end-product of heme catabolism produced by the gut microbiome. The structural difference between the two metabolites was the position of methyl and ethyl groups on the pyrrolidin-2-one moieties. We propose a mechanism of M18 and M19 generation from inavolisib and stercobilin whereby nucleophilic attack from the imidazole ring of inavolisib occurs to the bridging carbon of a stercobilin molecule. The proposed mechanism was supported by computational calculations of molecular orbitals and transition geometry. Significance Statement We report the characterization of two previously undescribed conjugates of the PI3K inhibitor inavolisib, generated by reaction with stercobilin, an end-product of heme catabolism produced by gut microbiome. These conjugates were confirmed by generating them using in vitro fecal homogenate incubation via non-enzymatic and possibly enzymatic reactions. Given the unique nature of the conjugate, it is plausible that it may have been overlooked with other small molecule drugs in prior studies.

Phytocannabinoid profile and potency of cannabis resin (hashish) of northwest Himalayas of India.

Cannabis is one of the most consumed illicit drugs and the potency of cannabis products is of note due to health-related concerns. Hand-rubbed hashish is the ancient technique of extracting psychoactive resin from cannabis plants and is practiced in the Indian Himalayas. This study establishes the cannabinoid profile and potency of hand-rubbed hashish collected from 20 regions of the northwest Himalayas. Fifty-eight hashish samples were analyzed using a validated high-performance liquid chromatography-diode array detection (HPLC-DAD) method. Ten cannabinoids were quantified including acidic (THCA & CBDA), and neutral compounds (CBDA, THCV, CBD, CBG, CBN, Δ9-THC, Δ8-THC, and CBC). The mean concentration (w/w%) of Δ9-THC is 26%; THCA is 15% and THCTotal is 40% is observed in the studied hashish samples. The majority (70%) of the hashish samples were categorized in chemotype I with the THC:CBD:CBN ratio of 91:3:4, and the remaining 30% were categorized under chemotype II with the ratio of 76:15:8. Diverse qualities of hashish are produced in the studied regions as per the seed, plant selection, and skills of manual rubbing, which results in potency variations. The average difference between the least and highest potent hand-rubbed hashish of a region is 27 w/w% (THCTotal). The other studied non-psychoactive cannabinoids have a mean w/w% of <5%, followed by 6% of CBDA. It is concluded that the cultivated and wild cannabis fields in the northwest Himalayas belong to the drug-type cannabis subspecies. Hand-rubbed hashish holds traditional significance and impacts the current policies of legislation.

Analysis of Raffinose Metabolism-Related Sugar Components in Seeds.

Raffinose family oligosaccharides (RFOs) are synthesized from sucrose and subsequent addition of galactose moieties which was provided by galactinol. Galactinol is synthesized from UDP-galactose and myo-inositol. RFOs accumulate at late stage of seed development and play important roles in seed longevity. RFOs are major components in seeds of many plant species. Here, we document a methodology for extraction and quantitative analysis of raffinose metabolism-related soluble sugars or the derivative alcohols in plant seeds. This protocol, based on high-performance liquid chromatography (HPLC), achieves the efficient separation and accurate quantification of sucrose, myo-inositol, galactinol, and raffinose within 25 min of retention time.

Trends in co-fractionation mass spectrometry: A new gold-standard in global protein interaction network discovery.

Co-fractionation mass spectrometry (CF-MS) uses biochemical fractionation to isolate and characterize macromolecular complexes from cellular lysates without the need for affinity tagging or capture. In recent years, this has emerged as a powerful technique for elucidating global protein-protein interaction networks in a wide variety of biospecimens. This review highlights the latest advancements in CF-MS experimental workflows including machine learning-guided analyses, for uncovering dynamic and high-resolution protein interaction landscapes with enhanced sensitivity, accuracy and throughput, enabling better biophysical characterization of endogenous protein complexes. By addressing challenges and emergent opportunities in the field, this review underscores the transformative potential of CF-MS in advancing our understanding of functional protein interaction networks in health and disease.

Spectrophotometric and chromatographic analysis of creatine:creatinine crystals in urine.

Creatinine is the end product of the catabolism of creatine and creatine phosphate. Creatine phosphate serves as a reservoir of high-energy phosphate, especially in skeletal and cardiac muscle. Besides typical known changes in serum and urinary creatinine concentrations, rare cases associated with changes in serum and urinary creatine levels have been described in the literature in humans. These cases are mostly linked to an excessive intake of creatine ethyl ester or creatine monohydrate, often resulting in increased urine creatinine concentrations. In addition, it is known that at such elevated creatinine concentrations, creatinine crystallisation may occur in the urine. Analysis of crystals and urinary concrements, often of heterogenous chemical composition, may provide diagnostic and therapeutic hints to the benefit of the patient. The aim of the present work was to analyze urine crystals of unclear composition with microscopic and spectroscopic techniques. On routine microscopic analysis of urine, a preliminary suspicion of uric acid or creatinine crystals was expressed. The crystals were of a cuboid shape and showed polarization effects in microscopy. The dried urine sample was whitish-orange in colour, odourless and dissolved well in water. Protein concentration in dry weight (DW) urine was about 0.3 mg/mg. The measured zinc content in the studied sample was approximately 660 µg/g DW sample and copper content was approximately 64 µg/g DW sample. A lead signal of around 10 µg/g DW sample was also observed. UV-Vis analysis showed a maximum creatine peak around 220 nm, compatible with the spectrum of creatinine with a maximum peak of 230 nm. Using HPLC technique, an extreme high ratio of creatine to creatinine of about 38 was measured, which led to the conclusion of the occurrence of rare creatine crystals in urine.

Comprehensive evaluation and screening of phytochemical compounds and their hypolipidemic activities of lotus leaf based on HPLC-Q-TOF-MS and spectral-effect analysis.

This study aimed to identify and quantify the primary components in lotus leaf and to explore the hypolipidemic components through spectral-effect relationships and chemometric methods. Utilizing a data-dependent acquisition-diagnostic fragment ion/characteristic neutral loss screening strategy (DFI-NLS), a reliable HPLC-Q-TOF-MS analysis was conducted, identifying 77 compounds, including 36 flavonoids, 21 alkaloids, 3 terpenoids, 11 organic acids, 4 phenols, 1 lignin and 1 unsaturated hydrocarbon. A straightforward HPLC-DAD method was developed for the simultaneous determination of seven major components in lotus leaf, and quercetin-3-O-glucuronide (Q3GA) was identified as the most abundant component. The HPLC fingerprints of 36 lotus leaf sample batches were assessed using chemometric approaches such as principal component analysis and hierarchical cluster analysis. The hypolipidemic effect of these samples was analyzed by measuring total cholesterol (TC) and total triglycerides (TG) levels in palmitic acid (PA) and oleic acid (OA)-induced lipid modeling in HepG-2 cells, employing partial least squares regression and grey relation analysis to investigate the spectral-effect relationship of the lotus leaf. The in vivo hypolipidemic effect of these compounds was assessed using an egg yolk powder-induced high-fat zebrafish model. The findings indicated that peak No.11 (Q3GA) in the chemical fingerprint was significantly associated with hypolipidemic activity, suggesting it as a potential hypolipidemic compound in lotus leaf. In summary, this study facilitates the exploration of the phytochemical compounds and their bioactive properties in the lotus leaf.

[Identification of chemical components in Qinggu San reference sample of classial prescription based on UHPLC-Q/Orbitrap HRMS combined with molecular network technology].

The method of ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry(UHPLC-Q/Orbitrap HRMS)combined with molecular network was developed in this study for rapidly analyzing the chemical components of the Qinggu San reference sample of classical prescription. Firstly, an ACQUITY UPLC BEH Shield RP_(18) column(2.1 mm×100 mm, 1.7 µm)was used, and acetonitrile and 0.1% formic acid were taken as the mobile phases for gradient elution. The flow rate was 0.4 mL·min~(-1), and the column temperature was 30 ℃. Under these conditions, the mass spectrum data were collected in both positive and negative ion modes of the heated electrospray ionization source. Subsequently, the mass spectrum data of the Qinggu San reference sample were uploaded to the Global Natural Products Social Molecular Network(GNPS)platform for calculation and analysis, and a visual molecular network was built with Cytoscape 3.8.2 software. On this basis, the chemical components of the Qinggu San reference sample were identified by fragmentation regularity of standard compounds, retention time, accurate relative molecular weight of HR-MS, characteristic fragment ions information, literature, and databases. Finally, a total of 105 chemical components were identified and speculated in the Qinggu San reference sample, including 19 iridoid glycosides, 23 flavonoids, 15 phenylpropanoids, 11 triterpene saponins, and 37 other components. Meanwhile, two of these components are potential new compounds. The method used in this study not only achieved rapid and accurate identification of chemical components in the Qinggu San reference sample and provided a scie-ntific basis for the study of pharmacological substances and quality control of Qinggu San compound preparations but also provided a refe-rence for the rapid identification of chemical components in traditional Chinese medicine compound preparations.

Identification of substituted variants of plastoquinone-9 as potent and specific photosynthetic inhibitors in cyanobacteria and plants.

The unprenylated benzoquinones 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone), 2-chloro-1,4-benzoquinone (CBQ), 2,6-dimethyl-1,4-benzoquinone (DMBQ), 2,6-dichloro-1,4-benzoquinone (DCBQ), and 2,6-dimethoxy-1,4-benzoquinone (DMOBQ) were tested as putative antimetabolites of plastoquinone-9, a vital electron and proton carrier of oxygenic phototrophs. Duroquinone and CBQ were the most effective at inhibiting the growth of the cyanobacterium Synechocystis sp. PCC 6803 either in photomixotrophic or photoautotrophic conditions. Duroquinone, a close structural analog of the photosynthetic inhibitor methyl-plastoquinone-9, was found to possess genuine bactericidal activity towards Synechocystis at a concentration as low as 10 µM, while at the same concentration CBQ acted only as a mild bacteriostat. In contrast, only duroquinone displayed marked cytotoxicity in axenically-grown Arabidopsis, resulting in damages to photosystem II and hindered net CO2 assimilation. Metabolite profiling targeted to photosynthetic cofactors and pigments indicated that in Arabidopsis duroquinone does not directly inhibit plastoquinone-9 biosynthesis. Taken together, these data indicate that duroquinone offers prospects as an algicide and herbicide.

Effect of crosslinker/porogen ratio on sponge-nested polymer monoliths for solid-phase extraction.

Polymer monoliths can be polymerised within different molds, but limited options are available for the preparation of free-standing polymer monoliths for analytical sample preparation, and in particular, solid-phase extraction (SPE). Commercial melamine-formaldehyde sponges can be used as supports for the preparation of polymer monoliths, due its flexibility, giving various shapes to monoliths. Herein, the crosslinker/porogen ratio of highly porous sponge-nested divinylbenzene (DVB) polymer monoliths has been evaluated. Monoliths prepared using different crosslinker/porogen ratios were applied to the extraction of bisphenol F, bisphenol A, bisphenol AF, and bisphenol B. Monoliths containing 50 wt % DVB and 50 wt % porogens presented the highest recovery of bisphenols. Under the optimised conditions, the developed method showed a linear range between 2.5 µg L-1 and 150 µg L-1 for BPA and BPAF, and between 5 µg L-1 and 150 µg L-1 for BPB and BPF. The limits of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10) ranged from 0.36 µg L-1 to 1.09 µg L-1, and from 1.20 µg L-1 to 3.65 µg L-1, respectively. The recoveries for spiked bisphenols (10 µg L-1) in tap water and water contained in a polycarbonate containers were between 82 % and 114 %.

An efficient microchip electromembrane extraction online with high-performance liquid chromatography for the measurement of nicotine in high consumption vegetables.

INTRODUCTION: Nicotine, a highly addictive substance, is naturally produced in the Solanaceae family of plants, particularly tobacco. The presence of nicotine in plant foods has adverse effects on the lungs, kidneys, heart, and reproductive system. OBJECTIVE: A novel three-phase microchip flat electromembrane coupled with online high-performance liquid chromatography (HPLC) was developed to analyze nicotine in tomato, mushroom, eggplant, bell pepper, and red pepper. METHODS: The microchip was connected to the HPLC in online mode. All effective variables were optimized to achieve the best extraction response. The use of electric potential and 2-nitrophenyl octyl ether -5% di(2-ethylhexyl) phosphate as a modified supported liquid membrane (SLM) increased the sensitivity and selectivity. RESULTS: The optimal extraction voltage, extraction time, and ion balance were 40 V, 10 min and 0, respectively. The dynamic linear range was 0.5-1000 ng g-1. The obtained recovery, relative standard deviation, and enrichment factor were 98%, 7%, and 35, respectively. The limits of detection 0.4 ng g-1 and the limits of quantification were obtained 1.3 ng g-1. The highest (105.0 ng g-1) and lowest (3.4 ng g-1) concentrations of nicotine were obtained for eggplant and tomato, respectively. CONCLUSION: Selective electromembrane extraction of nicotine from the donor phase to the acceptor phase was performed by optimizing the main variables influencing the method mechanism. The new channel design in this analytical system and online injection increased efficiency, stability, and repeatability. The results revealed that this method is capable for the efficient determination of trace amount of nicotine in edible vegetables.

Basal release of 6-cyanodopamine from rat isolated vas deferens and its role on the tissue contractility.

6-Cyanodopamine is a novel catecholamine released from rabbit isolated heart. However, it is not known whether this catecholamine presents any biological activity. Here, it was evaluated whether 6-cyanodopamine (6-CYD) is released from rat vas deferens and its effect on this tissue contractility. Basal release of 6-CYD, 6-nitrodopamine (6-ND), 6-bromodopamine, 6-nitrodopa, and 6-nitroadrenaline from vas deferens were quantified by LC-MS/MS. Electric-field stimulation (EFS) and concentration-response curves to noradrenaline, adrenaline, and dopamine of the rat isolated epididymal vas deferens (RIEVD) were performed in the absence and presence of 6-CYD and /or 6-ND. Expression of tyrosine hydroxylase was assessed by immunohistochemistry. The rat isolated vas deferens released significant amounts of both 6-CYD and 6-ND. The voltage-gated sodium channel blocker tetrodotoxin had no effect on the release of 6-CYD, but it virtually abolished 6-ND release. 6-CYD alone exhibited a negligible RIEVD contractile activity; however, at 10 nM, 6-CYD significantly potentiated the noradrenaline- and EFS-induced RIEVD contractions, whereas at 10 and 100 nM, it also significantly potentiated the adrenaline- and dopamine-induced contractions. The potentiation of noradrenaline- and adrenaline-induced contractions by 6-CYD was unaffected by tetrodotoxin. Co-incubation of 6-CYD (100 pM) with 6-ND (10 pM) caused a significant leftward shift and increased the maximal contractile responses to noradrenaline, even in the presence of tetrodotoxin. Immunohistochemistry revealed the presence of tyrosine hydroxylase in both epithelial cell cytoplasm of the mucosae and nerve fibers of RIEVD. The identification of epithelium-derived 6-CYD and its remarkable synergism with catecholamines indicate that epithelial cells may regulate vas deferens smooth muscle contractility.

Improved method for quantification of intact oxaliplatin by ultra high performance liquid chromatography-inductively coupled plasma mass spectrometry: Applications to clinical and speciation studies.

Interest is increasing in the use of different liquid chromatography techniques coupled online to mass spectrometry for the quantification of platinum anticancer drugs in human plasma to inform cancer chemotherapy. We developed, validated and studied the application of a method for quantification of intact oxaliplatin in human plasma using ultra high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (UHPLC-ICP-MS). Plasma samples were processed instantly after collection from patients to preserve oxaliplatin speciation by methanol-deproteinization, and storage of diluted supernatants (plasma:methanol 1:2 v/v) at -80 °C. UHPLC separation of intact oxaliplatin and internal standard (carboplatin) was achieved using a C18 column and linear gradient mobile phase (Mobile phase A: water-methanol (97:3 v/v), 0.075 mM sodium dodecyl sulfate, 9.79 nM thallium adjusted to pH 2.5 with trifluoromethanesulfonic acid; Mobile phase B: 100 % methanol (v/v)) with ICP-MS detection to monitor platinum and thallium at m/z 195 and 205, respectively. The limit of quantification was 50 nM in methanol-deproteinized diluted plasma (1:2 v/v). Linearity was established for calibration standards ranging from 50 to 500 nM made in methanol-deproteinized diluted plasma (1:2 v/v), and for dilution of higher concentration samples in blank matrix containing internal standard (final dilution 1:29 v/v). Intra-day and inter-day accuracy ranged from 96.8 to 103 % of nominal concentration and precision from 0.62 to 2.49 % coefficient of variation. Recovery was complete and a matrix effect confirmed the requirement for matrix-matched standards. Intact oxaliplatin was stable during storage for at least 473 days, and during analysis, in methanol-deproteinized diluted plasma (1:2 v/v). The method was applied to determining the plasma concentrations of intact oxaliplatin in patients undergoing cancer chemotherapy, and studies of oxaliplatin degradation in vitro. This improved method based on UHPLC-ICP-MS will allow more specific, efficient and reliable quantification of intact oxaliplatin in human plasma.

[Applications of high performance liquid chromatography-mass spectrometry in proteomics].

Proteomics profiling plays an important role in biomedical studies. Proteomics studies are much more complicated than genome research, mainly because of the complexity and diversity of proteomic samples. High performance liquid chromatography-mass spectrometry (HPLC-MS) is a fundamental tool in proteomics research owing to its high speed, resolution, and sensitivity. Proteomics research targets from the peptides and individual proteins to larger protein complexes, the molecular weight of which gradually increases, leading to sustained increases in structural and compositional complexity and alterations in molecular properties. Therefore, the selection of various separation strategies and stationary-phase parameters is crucial when dealing with the different targets in proteomics research for in-depth proteomics analysis. This article provides an overview of commonly used chromatographic-separation strategies in the laboratory, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), hydrophobic interaction chromatography (HIC), ion-exchange chromatography (IEC), and size-exclusion chromatography (SEC), as well as their applications and selectivity in the context of various biomacromolecules. At present, no single chromatographic or electrophoretic technology features the peak capacity required to resolve such complex mixtures into individual components. Multidimensional liquid chromatography (MDLC), which combines different orthogonal separation modes with MS, plays an important role in proteomics research. In the MDLC strategy, IEC, together with RPLC, remains the most widely used separation mode in proteomics analysis; other chromatographic methods are also frequently used for peptide/protein fractionation. MDLC technologies and their applications in a variety of proteomics analyses have undergone great development. Two strategies in MDLC separation systems are mainly used in proteomics profiling: the "bottom-up" approach and the "top-down" approach. The "shotgun" method is a typical "bottom-up" strategy that is based on the RPLC or MDLC separation of whole-protein-sample digests coupled with MS; it is an excellent technique for identifying a large number of proteins. "Top-down" analysis is based on the separation of intact proteins and provides their detailed molecular information; thus, this technique may be advantageous for analyzing the post-translational modifications (PTMs) of proteins. In this paper, the "bottom-up" "top-down" and protein-protein interaction (PPI) analyses of proteome samples are briefly reviewed. The diverse combinations of different chromatographic modes used to set up MDLC systems are described, and compatibility issues between mobile phases and analytes, between mobile phases and MS, and between mobile phases in different separation modes in multidimensional chromatography are analyzed. Novel developments in MDLC techniques, such as high-abundance protein depletion and chromatography arrays, are further discussed. In this review, the solutions proposed by researchers when encountering compatibility issues are emphasized. Moreover, the applications of HPLC-MS combined with various sample pretreatment methods in the study of exosomal and single-cell proteomics are examined. During exosome isolation, the combined use of ultracentrifugation and SEC can yield exosomes of higher purity. The use of SEC with ultra-large-pore-size packing materials (200 nm) enables the isolation of exosomal subgroups, and proteomics studies have revealed significant differences in protein composition and function between these subgroups. In the field of single-cell proteomics, researchers have addressed challenges related to reducing sample processing volumes, preventing sample loss, and avoiding contamination during sample preparation. Innovative methods and improvements, such as the utilization of capillaries for sample processing and microchips as platforms to minimize the contact area of the droplets, have been proposed. The integration of these techniques with HPLC-MS shows some progress. In summary, this article focuses on the recent advances in HPLC-MS technology for proteomics analysis and provides a comprehensive reference for future research in the field of proteomics.

Heme o Isolation and Analysis Using Heme o Synthase Heterologously Expressed in Escherichia coli.

Heme o is an Fe-porphyrin involved in the majority of aerobic respiration pathways found in all three domains of life. In eukaryotes and most aerobic prokaryotes, heme o functions solely as the precursor for the synthesis of heme a, a necessary cofactor for most heme-copper terminal oxidases. In some prokaryotes, such as Escherichia coli (E. coli), heme o can serve as a cofactor for heme-copper oxidases instead of heme a. Given its role as a key substrate or cofactor, purified heme o promises to be a valuable resource for the study of heme-copper oxidase assembly and activity. However, commercially available heme o is sold in limited quantities at a relatively high cost (compared to the prototypical heme b), making the use of heme o purchased from suppliers unfeasible for such studies. In this chapter, we present step-by-step methods both for heme o isolation from E. coli overexpressing heme o synthase and for HPLC analysis of cellular hemes (i.e., heme o and heme b).

Determination of eleven components in rat plasma by UPLC-MS/MS and GC-MS for pharmacokinetic studies after oral administration of Citri Reticulatae Pericarpium extract.

Citri Reticulatae Pericarpium (CRP) is used as common health-care food and traditional Chinese medicine (TCM), which exerts pharmacological effects, such as anti-cardiovascular, anti-tumor, anti-oxidant, anti-inflammatory, anti-virus, hepatoprotective, blood pressure-lowering and neuroprotective. In this study, reliable, and sensitive ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) methods were developed and validated for the determination of eleven active components in rat plasma after oral administration of the CRP extract. The results of this method exhibited that the specificity, linearity (r > 0.999), precision and accuracy (the coefficient of variation (CV) < 11.5 %), recovery (52.9-107.9 %), matrix effects (63.8-107.5 %), and stability (CV < 10.8 %) met all requirements for the quantitation of plasma samples. The pharmacokinetic results showed that the Tmax of flavone glycosides was less than 0.7 h, and that of polymethoxyflavones and volatile components were within 1-7 h. Meanwhile, the area-under-the-curve (AUC) and concentration maximum (Cmax) of hesperidin, nobiletin, tangeretin, and D-limonene were higher than those of the other components, suggesting that the plasma exposure levels of these constituents were higher in CRP. The present research lays a foundation for elucidating the therapeutic material basis and provides a reference for further scientific research and clinical application of CRP.

A combination of multiple LC-MS approaches for the comprehensive characterization of cysteine-linked ADCs.

Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.