A platform for qualitative and quantitative characterization of α-Gal and NeuGc at the oligosaccharide level.

Glycosylation modification serves as a pivotal quality attribute in glycoprotein-based therapeutics, emphasizing its role in drug safety and efficacy. Prior studies have underscored the potential immunogenic risks posed by the presence of galactose-α-1,3-galactose (α-Gal) and N-glycolylneuraminic acid (NeuGc) in glycoprotein formulations. This accentuates the imperative for developing robust qualitative and quantitative analytical methods to monitor these immunogenic epitopes, thereby ensuring drug safety. In the present investigation, α-Gal and NeuGc were accurately quantified using UPLC-FLR-MS/MS at the oligosaccharide level. Remarkably, α-Gal could be identified when the ion intensity ratio or the mass-to-charge ratio (m/z) of 528.19 to 366.14 exceeded 1. Concurrently, NeuGc and N-acetylneuraminic acid (NeuAc) could be unambiguously identified through their respective fragment ions at m/z 673.23 and m/z 657.23. Furthermore, relative quantification of α-Gal and NeuGc was achieved using fluorescence signals. This study introduces a sensitive and reliable analytical approach for the qualitative and quantitative assessment of α-Gal and NeuGc in glycoprotein pharmaceuticals. The methodology offers significant potential for application in process control and optimization of glycoprotein production, aimed at minimizing immunogenicity and enhancing product quality.

Selective and Sustainable Production of Sub-terminal Hydroxy Fatty Acids by a Self-Sufficient CYP102 Enzyme from Bacillus Amyloliquefaciens.

Enzymatic hydroxylation of fatty acids by Cytochrome P450s (CYPs) offers an eco-friendly route to hydroxy fatty acids (HFAs), high-value oleochemicals with various applications in materials industry and with potential as bioactive compounds. However, instability and poor regioselectivity of CYPs are their main drawbacks. A newly discovered self-sufficient CYP102 enzyme, BAMF0695 from Bacillus amyloliquefaciens DSM 7, exhibits preference for hydroxylation of sub-terminal positions (ω-1, ω-2, and ω-3) of fatty acids. Our studies show that BAMF0695 has a broad temperature optimum (over 70 % of maximal enzymatic activity retained between 20 to 50 °C) and is highly thermostable (T50 >50 °C), affording excellent adaptive compatibility for bioprocesses. We further demonstrate that BAMF0695 can utilize renewable microalgae lipid as a substrate feedstock for HFA production. Moreover, through extensive site-directed and site-saturation mutagenesis, we isolated variants with high regioselectivity, a rare property for CYPs that usually generate complex regioisomer mixtures. BAMF0695 mutants were able to generate a single HFA regiosiomer (ω-1 or ω-2) with selectivities from 75 % up to 91 %, using C12 to C18 fatty acids. Overall, our results demonstrate the potential of a recent CYP and its variants for sustainable and green production of high-value HFAs.

Universal protocol and standard-spiking strategy for profiling of host cell proteins in therapeutic growth hormone.

Liquid chromatography coupled to mass spectrometry (LC-MS) is widely used for host cell proteins (HCP) identification in antibody drug development because of its sensitivity, selectivity, and adaptability. However, LC-MS based identification of HCP in biotherapeutics produced from the prokaryotic Escherichia coli-derived growth hormone (GH) has rarely been reported. Herein, we developed a universal and powerful workflow by combining optimized sample preparation with one-dimension ultra-high performance LC-MS based shotgun proteomics to support HCP profiling in GH samples from downstream pools and the final product, which would be beneficial to direct the purification process development and compare the difference of impurity of different products for guiding the development of the biosimilar. A standard-spiking strategy was also developed to increase the depth of HCP identification. Spiking with standards enables additional identification of HCP species, which is promising for trace-level HCP analysis. Our universal and standard-spiking protocols would open an avenue for profiling HCP in biotherapeutics derived from prokaryotic host cells.

A tyrosine, histidine-selective bifunctional cross-linker for protein structure analysis.

Chemical cross-linking mass spectrometry (XL-MS) significantly contributes to the analysis of protein structures and the elucidation of protein-protein interactions. Currently available cross-linkers mainly target N-terminus, lysine, glutamate, aspartate, and cysteine residues in protein. Herein, a bifunctional cross-linker, named [4,4'-(disulfanediylbis(ethane-2,1-diyl)) bis(1-methyl-1,2,4-triazolidine-3,5-dione)] (DBMT) has been designed and characterized aiming to extremely expand the application of XL-MS approach. DBMT is capable of selectively targeting tyrosine residue in protein via an electrochemical click reaction, and/or targeting histidine residue in protein in the presence of 1O2 generated under photocatalytic reaction. A novel cross-linking strategy based on this cross-linker has been developed and demonstrated using model proteins, which provides a complementary XL-MS tool analyzing protein structure, protein complexes, protein-protein interactions, and even protein dynamics.

PEGylated Recombinant Human Growth Hormone Jintrolong® Exhibits Good Long-Term Safety in Cynomolgus Monkeys and Human Pediatric Growth Hormone Deficiency Patients.

Jintrolong® is a long-acting PEGylated recombinant human growth hormone (PEG-rhGH) developed for weekly injection in patients with pediatric growth hormone deficiency (PGHD). Although PEG modification of therapeutic proteins is generally considered safe, concerns persist about the potential for adverse vacuolation in tissues with long-term exposure to PEG-included therapies, particularly in children. We assessed the safety of Jintrolong® in cynomolgus monkeys with an examination of vacuolation in the brain choroid plexus (CP) and reported long-term clinical safety data obtained from children with PGHD. The toxicity of Jintrolong® was assessed following the 52-week administration with doses at 0.3, 1, or 3 mg/kg/week. The levels of vacuolation of CP in animals were dose-dependent and at least partially reversible after a 104- or 157-week recovery period. Vacuolation in the CP epithelium did not lead to obvious subcellular structural or cell functional abnormalities. Compared with the clinical dose of 0.2 mg/kg/week Jintrolong® in PGHD patients, exposure in monkeys under NOAEL 3 mg/kg/week exhibited safety margins greater than 120.5, the predicted minimum dose to induce vacuolation in monkeys is equivalent to 1.29 mg/kg/week in humans, which is 6.45-fold higher than the clinical dose. The safety data acquired in clinical trials for Jintrolong® were also analyzed, which included phase III (360 patients), phase IV (3,000 patients) of 26-week treatment, and a follow-up study with treatment lasting for 3 years. There was no statistically significant difference in the incidence of adverse reactions between the Jintrolong® group and the daily rhGH control group (no PEG), and no new adverse effects (AE) were observed in the Jintrolong® group at the clinical therapeutic dose of 0.2 mg/kg/week.

Legionella pneumophila regulates host cell motility by targeting Phldb2 with a 14-3-3ζ-dependent protease effector.

The cytoskeleton network of eukaryotic cells is essential for diverse cellular processes, including vesicle trafficking, cell motility, and immunity, thus is a common target for bacterial virulence factors. A number of effectors from the bacterial pathogen Legionella pneumophila have been shown to modulate the function of host actin cytoskeleton to construct the Legionella-containing vacuole (LCV) permissive for its intracellular replication. In this study, we found that the Dot/Icm effector Lem8 (Lpg1290) is a protease whose activity is catalyzed by a Cys-His-Asp motif known to be associated with diverse biochemical activities. Intriguingly, we found that Lem8 interacts with the host regulatory protein 14-3-3ζ, which activates its protease activity. Furthermore, Lem8 undergoes self-cleavage in a process that requires 14-3-3ζ. We identified the Pleckstrin homology-like domain-containing protein Phldb2 involved in cytoskeleton organization as a target of Lem8 and demonstrated that Lem8 plays a role in the inhibition of host cell migration by attacking Phldb2.

Transferrin-conjugated liposomes loaded with carnosic acid inhibit liver cancer growth by inducing mitochondria-mediated apoptosis.

Our previous studies have proven that carnosic acid (CA) induces apoptosis of liver cancer cells. However, the poor chemical properties of CA limit its in vivo anti-cancer effects. In this study, CA was loaded into liposomes (LP-CA), and LP-CA was further conjugated with transferrin (Tf-LP-CA) to overcome the shortcomings of poor solubility and absorption at the lesion site. In HepG2 and SMMC-7721 cells, compared with CA and LP-CA, more Tf-LP-CA was absorbed by liver cancer cells, which induced higher levels of apoptosis and reduced the mitochondrial membrane potential more effectively. In HepG2- and SMMC-7721-xenotransplanted mice, Tf-LP-CA inhibited tumor growth with no cytotoxicity to the liver, spleen, or kidney. Furthermore, compared with CA and LP-CA, Tf-LP-CA targeted the tumor site more effectively, enhanced the expressions of cleaved poly(ADP-ribose) polymerase, and Caspase-3 and -9, and regulated the expression levels of B-cell lymphoma 2 (Bcl2) family members in the tumor tissues. Tf-LP-CA was taken up by tumor cells and targeted at tumor tissues, ensuring the precise delivery of CA, which further promoted mitochondria-mediated intrinsic apoptosis in the liver cancer cells. These results provide evidence for the clinical application of the Tf-LP-based CA drug delivery system for liver cancer.

Wan-Nian-Qing, a Herbal Composite Prescription, Suppresses the Progression of Liver Cancer in Mice by Regulating Immune Response.

The Wan-Nian-Qing prescription (WNQP), an herbal composite containing Ornithogalum caudatum, has been used in China for several years for cancer treatment. However, the mechanism of its pharmacological action against liver cancer is not clear. This study aimed to investigate the role of WNQP in inhibiting tumor growth in hepatocellular carcinoma model mice and determine its mechanism of action. We established HepG2- and SMMC-7721-xenografted tumor models in nude mice and BALB/c mice. The mice were administered WNQP for 2 weeks. The bodyweight of each mouse was monitored every day, and the tumor size was measured using vernier caliper before each round of WNQP administration. After the last dose, mice were sacrificed. The tumors were removed, lysed, and subjected to proteome profiling, enzyme-linked immunosorbent assay, and western blotting. The liver, spleen, and kidney were collected for histopathological examination. The effects of WNQP against liver cancer were first systematically confirmed in HepG2- and SMMC-7721-xenografted nude mice and BALB/c mice models. WNQP inhibited tumor growth, but failed to affect bodyweight and organ structures (liver and spleen), confirming that it was safe to use in mice. In BALB/c mice, WNQP regulated immune function, inferred from the modulation of immune-related cytokines such as interleukins, interferon, tumor necrosis factors, and chemokines. Further results confirmed that this regulation occurred via the regulatory effects of WNQP on Nrf2 signaling. WNQP can inhibit the growth of HepG2- and SMMC-7721-xenografted tumors in nude mice and BALB/c mice. This effect manifests at least partially through immunomodulation mediated apoptosis.

Influence of drying and calcination temperatures for Ce-Cu-Al trimetallic composite catalyst on simultaneous removal H2S and PH3: Experimental and DFT studies.

This work explored the influences of the drying and calcination temperatures on a Ce-Cu-Al trimetallic composite catalyst for the simultaneous removal of H2S and PH3. The effects of both temperatures on the structural features and activity were examined. The density functional theory method was used to calculate adsorption energies and further analyze their adsorption behavior on different slabs. Experiments revealed suitable drying and calcination temperatures to be 60 and 500°C, respectively. The capacity reached 323.8 and 288.1 mg/g. Adjusting drying temperature to 60°C is more inclined to form larger and structured grains of CuO. Rising calcinating temperature to 500°C could increase the grain size and redox capacity of CuO to promote performance. Higher temperatures would destroy the surface structure and lead to a crystal phase transformation, which was that the CuO and Al2O3 were gradually recombined into CuAl2O4 with a spinel structure. The exposed crystal planes of surficial CuO and CuAl2O4 were determined according to characterization results. Calculation results showed that, compared with CuO (111), H2S and PH3 have weaker adsorption strength on CuAl2O4 (100) which is not conducive to their adsorption and removal.

The anti-carcinogenesis properties of erianin in the modulation of oxidative stress-mediated apoptosis and immune response in liver cancer.

In this study, erianin was found to reduce the viability of cancer cells, inhibit their proliferation and migration, induce G2/M phase arrest, enhance cancer cell apoptosis, promote an increase in levels of intracellular reactive oxygen species and a decrease in mitochondrial membrane potential, and regulate the expression levels of anti- and pro-apoptosis-related proteins in HepG2 and SMMC-7721 cells. Erianin inhibited tumor growth in HepG2- and SMMC-7721-xenograft tumor nude mouse models, reduced the expression levels of anti-apoptosis proteins and enhanced the expression levels of pro-apoptosis proteins in tumor tissues. Erianin inhibited tumor growth in immunosuppressed BALB/c mice bearing heterotopic tumors. Among 111 types of cytokines detected in proteome profiling of tumor tissues, erianin substantially influenced levels of 38 types of cytokines in HepG2-xenografted tumors and of 15 types of cytokines in SMMC-7721-xenografted tumors, most of which are related to immune functions. Erianin strongly affected the serum levels of cytokines, and regulated the activation of nuclear factor-kappa B (NF-κB), and the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream proteins in spleen. The anti-liver cancer properties of erianin were found to be related mostly to its modulation of oxidative stress-mediated mitochondrial apoptosis and immune response.

Characterization of recombinant monoclonal antibody charge variants using WCX chromatography, icIEF and LC-MS/MS.

Charge heterogeneity is an important aspect of research into the development of monoclonal antibody drugs. In the present study, charge variants were separated into four fractions using weak cation exchange chromatography and were thoroughly analyzed using liquid chromatography-mass spectrometry at multiple levels. Molecular weight analysis of intact antibody and subunits confirmed the presence of heavy-chain leader sequences, light-chain leader sequences, dehydration, and cysteinylation. Peptide mapping of the fractions using different enzymes further localized the modified sites. Modified proportions identified at peptide level were compared with the purity detected by imaged capillary isoelectric focusing, the results showed that basic variant 1 consisted of cysteinylation and dehydration of asparagine, and basic variant 2 fully accounted for the N-terminal leader sequence of the heavy chain. About 14.8% of the acidic variant can be explained by N-terminal leader sequences in the light chain, and 18% of the acidic variant was demonstrated to be deamidation of asparagine in the heavy chain. There was approximately 54.2% of the acidic variant still cannot be explained. It was hypothesized that those acidic variants that have not yet been identified are an ensemble of molecules with slight molecular weight differences or the same molecular weight but different structures.

Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors.

Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

Cordyceps militaris induces tumor cell death via the caspase­dependent mitochondrial pathway in HepG2 and MCF­7 cells.

Cordyceps militaris (CM), an entomopathogenic fungus belonging to the class ascomycetes, possesses various pharmacological activities, including cytotoxic effects, on various types of human tumor cells. The present study investigated the anti­hepatocellular carcinoma (HCC) and anti­breast cancer effects of CM in in vitro and in vivo models. CM aqueous extract reduced cell viability, suppressed cell proliferation, inhibited cell migration ability, caused the over-release of lactate dehydrogenase, induced mitochondrial dysfunction and enhanced apoptotic rates in MCF­7 and HepG2 cells. The expression levels of cleaved poly (ADP ribose) polymerase and caspase­3, biomarkers of apoptosis, were increased following treatment with CM aqueous extract for 24 h. Furthermore, in the MCF­7 and HepG2 cells, enhanced levels of B cell­associated X protein and cleaved caspase­8 were observed in the CM­treated cells. Finally, the antitumor activities of CM in HCC and breast cancer were also confirmed in MCF­7­ and HepG2­xengraft nude mice models. Collectively, the data obtained in the present study suggested that the cytotoxic effects of CM aqueous extract on HCC and breast cancer are associated with the caspase­dependent mitochondrial pathway.

Studies on the Antidiabetic and Antinephritic Activities of Paecilomyces hepiali Water Extract in Diet-Streptozotocin-Induced Diabetic Sprague Dawley Rats.

Paecilomyces hepiali is a fungus widely used in Asian countries for various potential pharmacological activities. The present study aims to evaluate the antidiabetic and antinephritic effects of the Paecilomyces hepiali mycelium water extract (PHC) in diabetic rat, which is established by eight-week high-fat diet administration followed by one-week tail intravenous injection of 25 mg/kg streptozotocin (STZ). After four-week 0.12 g/kg metformin and PHC at doses of 0.08, 0.4, and 2.0 g/kg treatment, an increment of body weight, a decrement of plasma glucose, low levels of total cholesterol, and low density lipoprotein cholesterol in diabetic rats were observed. PHC promotes glucose metabolism by enhancing insulin, pyruvate kinase activity, and increasing the synthesis of glycogen. PHC normalized the disturbed levels of superoxide dismutase, methane dicarboxylic aldehyde, and glutathione peroxidase in kidney. The inhibitory effects on the levels of interleukin-2, interleukin-6, interleukin-10, and tumor necrosis factor-α in serum and kidney revealed the protection of PHC against diabetic nephropathy. Compared with nontreated diabetic rats, four-week PHC treatment resulted in a decrement on nuclear factor kappa B expression in kidney. These results show that Paecilomyces hepiali possesses antidiabetic and antinephritic effects which are related to the modulation of nuclear factor kappa B activity.

Cordyceps militaris fruit body extract ameliorates membranous glomerulonephritis by attenuating oxidative stress and renal inflammation via the NF-κB pathway.

Membranous glomerulonephritis (MGN) is a common pathogenesis of nephritic syndrome in adult patients. Nuclear factor kappa B (NF-κB) serves as the main transcription factor for the inflammatory response mediated nephropathy. Cordyceps militaris, containing various pharmacological components, has been used as a kind of crude drug and folk tonic food for improving immunity and reducing inflammation. The current study aims to investigate the renoprotective activity of Cordyceps militaris aqueous extract (CM) in the cationic bovine serum albumin (C-BSA)-induced rat model of membranous glomerulonephritis. Significant renal dysfunction was observed in MGN rats; comparatively, 4-week CM administration strongly decreased the levels of 24 h urine protein, total cholesterol, triglyceride, blood urea nitrogen and serum creatinine, and increased the levels of serum albumin and total serum protein. Strikingly, recovery of the kidney histological architecture was noted in CM-treated MGN rats. A significant improvement in the glutathione peroxidase and superoxide dismutase levels, and a reduced malondialdehyde concentration were observed in the serum and kidney of CM-treated rats. Altered levels of inflammatory cytokines including interleukins, monocyte chemoattractant protein-1, intercellular adhesion molecule 1, vascular adhesion molecule 1, tumor necrosis factor-α, 6-keto-prostaglandin F1α, and nuclear transcriptional factor subunit NF-κB p65 reverted to normal levels upon treatment with CM. The present data suggest that CM protects rats against membranous glomerulonephritis via the normalization of NF-κB activity, thereby inhibiting oxidative damage and reducing inflammatory cytokine levels, which further provide experimental evidence in support of the clinical use of CM as an effective renoprotective agent.

Studies on the Antifatigue Activities of Cordyceps militaris Fruit Body Extract in Mouse Model.

Cordyceps militaris has been used extensively as a crude drug and a folk tonic food in East Asia due to its various pharmacological activities. Our study aims to investigate the effect of Cordyceps militaris fruit body extract (CM) on antifatigue in mouse model. Two week CM administration significantly delayed fatigue phenomenon which is confirmed via rotating rod test, forced swimming test and forced running test. Compared to nontreated mouse, CM administration increased ATP levels and antioxidative enzymes activity and reduced the levels of lactic acid, lactic dehydrogenase, malondialdehyde, and reactive oxygen species. Further data suggests that CM-induced fatigue recovery is mainly through activating 5'-AMP-activated protein kinase (AMPK) and protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathways and regulating serum hormone level. Moreover, CM-enhanced the phosphorylation of AMPK contributes to its antioxidant effect. Our data provides experimental evidence in supporting clinical use of CM as an effective agent against fatigue.

A liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of escin Ia and escin Ib in human plasma: application to a pharmacokinetic study after intravenous administration.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantification of escin Ia and escin Ib in human plasma. After a solid-phase extraction (SPE), the analytes were separated on a Zorbax Extend C(18) column by isocratic elution with a mobile phase of methanol-acetonitrile-10 mm ammonium acetate (27:27:46, v/v/v) at a flow rate of 1.0 mL/min and analyzed by mass spectrometry in the positive ion multiple reaction monitoring mode. The precursor to product ion transitions of m/z 1131.8 → 807.6 was used to quantify escin Ia and escin Ib. Good linearity was achieved over a wide range of 2.00-900 ng/mL for escin Ia and 1.50-662 ng/mL for escin Ib. The intra- and inter-day precisions (as relative standard deviation) were less than 11% for each QC level of escin Ia and escin Ib. The accuracies (as relative error) were within ±5.27% for escin Ia and within ±4.07% for escin Ib. The method was successfully employed in a pharmacokinetic study after a single intravenous infusion administration of sodium aescinate injection containing 10 mg escin to each of the 10 healthy volunteers.

Simultaneous analysis of isomers of escin saponins in human plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study after oral administration.

A rapid and sensitive bioassay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of four isomeric escin saponins (escin Ia, escin Ib, isoescin Ia and isoescin Ib) in human plasma has been developed and validated. Sample preparation of plasma after addition of telmisartan as internal standard (I.S.) involved solid-phase extraction (SPE) on C18 cartridges. Separation was based on reversed phase chromatography using gradient elution with methanol-acetonitrile (50:50, v/v) and 10 mM ammonium acetate solution (pH 6.8). MS/MS detection in the positive ion mode used multiple reaction monitoring of the transition at m/z 1113.8-->807.6. Stability issues with the four saponins required the addition of formic acid to plasma samples prior to storage at -80 degrees C and analysis within 30 days. The method was linear at concentrations up to 10 ng/mL with correlation coefficients>0.996 for all analytes. The lower limit of quantitation (LLOQ) for all four saponins was 33 pg/mL. Intra- and inter-day precisions (as relative standard deviation) were all <15% and accuracies (as relative error) in the range -5.3% to 6.1%. The method was successfully applied to a pharmacokinetic study of escins in healthy volunteers after oral administration of sodium aescinate tablets containing 60 mg escin saponins.

Rapid and sensitive LC-MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma.

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh(2), were extracted from plasma by liquid-liquid extraction and separated on a Zorbax extend C(18) analytical column using methanol-acetonitrile-10mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1-100.0ng/ml with a limit of detection of 0.05ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25mg capsule.

Quantitation of bergenin in human plasma by liquid chromatography/tandem mass spectrometry.

This paper reports the development and validation of an assay for quantitation of bergenin in human plasma using liquid chromatography/tandem mass spectrometry (LC-MS/MS). Bergenin and the internal standard (I.S.), 5-bromo-2,4(1H,3H)-pyrimidinedione (5-BrU), were separated by reversed phase HPLC and quantitated by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the negative ion mode. The most intense [M-H](-) MRM transition of bergenin at m/z 326.9-->312.3 was used for quantitation and the transition at m/z 188.9-->42.2 was used to monitor 5-BrU. Stability issues with bergenin required the addition of ascorbic acid to plasma samples prior to storage and analysis within 10 days storage at -80 degrees C. The method was linear in the range 3-1000 ng/mL with intra- and inter-day precision of 3.94-5.96 and 1.62-8.31%, respectively, and accuracy <2.33%. The assay was successfully applied to a pharmacokinetic study in healthy volunteers after administration of a single 250 mg oral dose.