Accurate LC Research Articles

Wide Remote-Range and Accurate Wireless LC Temperature-Humidity Sensor Enabled by Efficient Mutual Interference Mitigation.

Inductor-capacitor wireless integrated sensors (LCWISs) featuring untethered and multitarget measurements are promising in health monitoring and human-machine interfaces. However, the lack of a profound understanding of the internal interference hinders the design of the LCWIS, which has a wide remote sensing range and high accuracy. Herein, a mutually exclusive effect of the mutual inductance interferences in LCWIS was revealed and quantified, enabling a design with a wide range of remote sensing (working distance comparable to the single-target device, working radius: 4 mm) and 16% reduced area. As a key to accurate multitarget measurement, a quantified target interference model based on interference decomposition was proposed to understand the target interferences, providing profound guidance for the design of ultra-accurate LCWIS. As a proof, we designed a cellulose-polyacrylate-cellulose LCWIS (CPC-LCWIS) with ultrahigh accuracies (∼1.2% RH and ∼0.18 °C) beyond commercial wired gauges. The CPC-LCWIS with full-coil sensing structures achieved exceptionally high sensitivities (0.36 MHz/°C and 0.25 MHz/% RH). The CPC-LCWIS was validated for health monitoring and human-machine interfaces. The concept studied in this work provides profound guidance for designing a high-performance flexible LCWIS for advanced wearable electronics.

Development, validation and application of a capillary microsampling LC–MS/MS method for the quantification of BIIB131 (SMTP-7) in rat plasma

Capillary microsampling (CMS) is a technique that can significantly reduce the blood collection volume compared to conventional sampling methods, and thus is much preferred for studies in rats and mice. BIIB131 (SMTP-7) is a novel thrombolytic drug candidate currently under Phase 2 clinical development for the treatment of acute ischemic stroke. To support the safety studies in rats, an accurate and reliable CMS LC–MS/MS assay for the quantification of BIIB131 in rat plasma was developed and validated. This method utilized stable-isotope labeled [13C515N2]-BIIB131 as the internal standard. The samples were extracted using acid-assisted liquid–liquid extraction with methyl tert-butyl ether (MTBE) and formic acid. The chromatographic separation was achieved on an ACE Excel 3 Super C18 analytical column (2.1 mm × 50 mm, 3.0 µm) using a gradient elution. The mass spectrometric detection of BIIB131 and its internal standard was achieved using positive ion electrospray multiple reaction monitoring (MRM). The standard curve ranged from 0.50 to 300 ng/mL for BIIB131 and was fitted to a 1/x2 weighted linear regression model. For regular QCs, the intra-assay precision was 1.7–6.1 % CV, the inter-assay precision was 2.7–11.0 % CV, and the intra-assay and inter-assay accuracy (%Bias) were –20.0–10.6 % and –7.8–6.3 %, respectively. For CMS QCs, the intra-assay and inter-assay precision were 2.2–13.6 % and 6.7–12.9 % CV, and the intra-assay and inter-assay accuracy (%Bias) were –13.2–15.0 % and –7.8–4.2 %, respectively. The validated CMS LC–MS/MS method has been successfully applied to a safety study in rats.

Development and validation of LC–MS/MS methods for the determination of EVT201 and its five metabolites in human urine: Application to a mass balance study

In this study, two simple and accurate LC–MS/MS methods were firstly developed and validated to quantify EVT201, a new partial GABAA receptor agonist used for the treatment of insomnia, and its metabolites comprising M1, M2, M3, M4 and M6 in human urine. The analytes in urine samples were determined after simple dilution, and ideal chromatographic separations were obtained on C18 columns using gradient elution. The assays were performed in MRM mode on AB QTRAP 5500 tandem mass spectrometry (ESI+). The concentration ranges (ng/mL) of analytes in human urine were as follows: EVT201, 1.00 to 36.0; M1, 1.40 to 308; M2, 2.00 to 72.0; M3, 5.00 to 1100; M4, 2.00 to 300; and M6, 2.80 to 420. The methods were fully validated including selectivity, carryover, matrix effect, recovery, linearity, accuracy, precision, dilution integrity and stability, and acceptable criteria were obtained. The methods were successfully applied to a mass balance study of EVT201. The results showed that the total cumulative urinary excretion rate of EVT201 and its five metabolites was 74.25 ± 6.50%, which suggested that EVT201 had high oral bioavailability, and urinary elimination was its major excretion pathway in human.

Analytical Method Development of Benzisothiazolinone, a Biocide, Using LC-MS/MS and a Pharmacokinetic Application in Rat Biological Matrices.

Benzisothiazolinone (BIT), a biocide widely used as a preservative in household cleaning and personal care products, is cytotoxic to lung cells and a known skin allergen in humans, which highlights the importance of assessing its toxicity and pharmacokinetics. In this study, a simple, sensitive, and accurate LC-MS/MS method for the quantification of BIT in rat plasma, urine, or tissue homogenates (50 μL) using phenacetin as an internal standard was developed and validated. Samples were extracted with ethyl acetate and separated using a Kinetex phenyl-hexyl column (100 × 2.1 mm, 2.6 μm) with isocratic 0.1% formic acid in methanol and distilled water over a run time of 6 min. Positive electrospray ionization with multiple reaction monitoring transitions of m/z 152.2 > 134.1 for BIT and 180.2 > 110.1 for phenacetin was used for quantification. This assay achieved good linearity in the calibration ranges of 2-2000 ng/mL (plasma and urine) and 10-1000 ng/mL (tissue homogenates), with r ≥ 0.9929. All validation parameters met the acceptance criteria. BIT pharmacokinetics was evaluated via an intravenous and dermal application. This is the first study that evaluated BIT pharmacokinetics in rats, providing insights into the relationship between BIT exposure and toxicity and a basis for future risk assessment studies in humans.

SIMULTANEOUS LC-MS/MS QUANTIFICATION METHOD FOR REMOGLIFLOZIN ETABONATE AND METFORMIN IN PLASMA SAMPLES: APPLICATION OF KINETIC STUDY IN RABBITS

A new accurate LC–MS/MS methodology was needed for the simultaneous quantitation of remogliflozin etabonate and metformin in plasma samples. Chromatography was executed with BDS-Hyoersil, C18 (50mm×4.60mm, 5.0µ) reverse phasic analytical stationary system with movable solvent comprising of 0.10% HCOOH, methyl alcohol, and acetonitrile in the fraction of 10:45:45. Multiple reaction monitoring exhibits ionic conversions at m/e 130.14 →85.08, m/e 523.26 → 271.18 and 586.19 → 57.07 for Metformin (MTF), Remogliflozin etabonate (RME), Fosamprenavir internal standard (IS) correspondingly. Method recovery outcomes were not less than 90%, and findings of accuracy values were within the relative error of ±6.0%. MTF has a mean Tmax of 5.62 ± 0.22. AUC0last was 374.86 ± 9.889 and Cmax was 1.89 ± 1.23 for the sample. RME has a mean Tmax of 52.99 ± 0.12. AUC0last was 265.83± 9.81 and Cmax was 41.13 ± 0.97 for the sample.

Hyperactive DNA Cutting for Unbiased UHPLC-MS/MS Quantification of Epigenetic DNA Marks by Engineering DNase I Mutants.

Epigenetic DNA modifications, such as 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine, are associated with a variety of diseases and potential biomarkers for cancer diagnosis and therapy. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays are considered to be the gold standard for qualitative and quantitative detection of DNA modifications. DNA digestion for converting long DNA polymer into 2'-deoxynucleosides is an important preprocessing step to achieve sensitive and accurate LC-MS/MS quantification. Here, we showed that, as stimulated by divalent metal ions, Mg2+ and Mn2+, the engineered human DNase I Q9R:E13R:N74K mutant can efficiently digest DNA in the presence of monovalent metal ions at a high concentration (e.g., 1 M NaCl), showing hyperactivity on DNA cutting. We also found that the engineered DNase I mutants display exceptional DNA-cutting activity over a wider pH range (5.5-9.5). Due to their hyperactivity and high salt tolerance, the engineered DNase I mutants cut DNA 5mC and dC efficiently. Benefitting from this DNA-cutting hyperactivity, we demonstrated an LC-MS/MS assay for unbiased and accurate quantification of DNA 5mC.

A Parameter Extraction Method for LC Circuit of DB-BPF Based on Fully Connected Network

A parameter extraction method for the LC circuit of a dual-band bandpass filter (DB-BPF) based on the fully connected network is proposed in this article. The network learning process consists of two stages: 1) pretraining and 2) fine-tuning. The network after pretraining has the ability to output the inaccurate values of LC circuit parameters with the desired <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">${S}$ </tex-math></inline-formula> -parameters (obtained according to the filter design requirements) as input. The precise LC circuit parameters can be obtained from the network during the subsequent fine-tuning process. This parameter extraction method can be applied to LC circuits of various bandpass filters. In addition to the amplitude of S-parameter, the phase of S-parameter is also used as learning data for the pretraining network. Comparative experiments show that the most accurate LC circuit parameters can be predicted using the network trained with phase information compared with the network trained without phase information. Two 7-order narrowband and two 9-order wideband DB-BPFs with four transmission zeros at different center frequencies are designed to demonstrate the validity of the proposed method, and the experimental results show that the proposed method is valid and effective.

Simultaneous and enantiospecific quantification of primaquine and carboxyprimaquine in human plasma using liquid chromatography-tandem mass spectrometry

BackgroundThe enantiomers of the 8-aminoquinoline anti-malarial primaquine have different pharmacological properties. Development of an analytical method for simultaneous quantification of the enantiomers of primaquine and its metabolite, carboxyprimaquine, will support clinical pharmacometric assessments.MethodsA simple and sensitive method consisting of liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) was developed for simultaneous and enantiospecific determination of primaquine and its metabolite, carboxyprimaquine, in human plasma. Stable isotopes were used as internal standards to compensate for potential interference and matrix effects. Plasma samples (100 µL) were precipitated with 1% formic acid in acetonitrile followed by phospholipid removal solid phase extraction. Primaquine and carboxyprimaquine enantiomers were separated on a Chiralcel OD-3R (150 mm × 4.6 mm; I.D. 3 μm) column using a LC gradient mode. For separation of racemic primaquine and carboxyprimaquine, the LC method was modified and validated using a reverse phase column (Hypersil Gold 100 mm × 4.6 mm; I.D. 3 µm) and a mobile phase composed of 10 mM ammonium acetate buffer, pH 3.5 and acetonitrile in the isocratic mode. Method validation was performed according to regulatory guidelines.ResultsThe calibration range was set to 0.571–260 ng/mL and 2.44–2,500 ng/mL for primaquine and carboxyprimaquine enantiomers, respectively, resulting in a correlation coefficient (r2) ≥ 0.0998 for all calibration curves. The intra- and inter-day assay precisions were < 10% and the accuracy was between 94.7 to 103% for all enantiomers of primaquine and carboxyprimaquine. The enantiospecific method was also modified and validated to quantify racemic primaquine and carboxyprimaquine, reducing the total run time from 30 to 8 min. The inter-, intra-day assay precision of the racemic quantification method was < 15%. The absolute recoveries of primaquine and carboxyprimaquine were between 70 and 80%. Stability was demonstrated for up to 2 years in − 80 °C. Both the enantiomeric and racemic LC–MS/MS methods were successfully implemented in pharmacokinetic studies in healthy volunteers.ConclusionsSimple, sensitive and accurate LC–MS/MS methods for the quantification of enantiomeric and racemic primaquine and carboxyprimaquine in human plasma were validated successfully and implemented in clinical routine drug analysis.

Identification and Quantification of Phthalates in Drinking, River and Lake Water using Solid phase extraction followed by UHPLC-MS

Phthalates are endocrine disruptive compounds that are commonly employed in everyday consumer items. Anti-androgenic action is the possible negative consequences are associated with phthalates and hence there is growing worry regarding human exposure to phthalates. It is critical to have a validated analytical approach that can measure trace quantities of phthalate metabolites in drinking water when investigating environmental exposure to phthalates. In this investigation, we developed and validated an accurate, sensitive and robust LC–MS technique to concurrently detect six phthalates in drinking water samples: dimethyl (DMP), diethyl (DEP), dipentyl (DPrP), diisobutyl (DIBP), dibutyl (DBP) and dioctyl (DOP). These analytes were quantified by using LC– MS system with electrospray ionization. The validated approach has been utilised effectively in measuring phthalate exposure in drinking water samples and water samples collected at various locations. Packaged drinking water samples separated into two groups, room temperature samples (15 days, 30 days, 60 days, 120 days, 150 days and 180 days) and heated samples at 50 oC with similar interval samples.

Establishment and validation of an SIL‐IS LC–MS/MS method for the determination of ibuprofen in human plasma and its pharmacokinetic study

AbstractIn this work, we developed and validated a highly sensitive, rapid and stable LC–MS/MS method for the determination of ibuprofen in human plasma with ibuprofen‐d3 as a stable isotopically labeled internal standard (SIL‐IS). Human plasma samples were prepared by one‐step protein precipitation. The chromatographic separation was achieved on a Poroshell 120 EC‐C18 (2.1 × 50 mm, 2.7 μm). Aqueous solution (containing 0.05% acetic acid and 5 mm NH4Ac) and methanol were selected as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in negative ion mode. Multiple reaction monitoring mode was used for quantification using target fragment ions m/z 205.0 → 161.1 for ibuprofen and m/z 208.0 → 164.0 for SIL‐IS, respectively. This method exhibited a linear range of 0.05–36 μg/ml for ibuprofen with correlation coefficient &gt;0.99. Mean recoveries of ibuprofen in human plasma ranged from 78.4 to 80.9%. The RSD of intra‐ and inter‐day precision were both &lt; 5%. The accuracy was between 88.2 and 103.67%. The matrix effect was negligible in human plasma, including lipidemia and hemolytic plasma. A simple, efficient and accurate LC–MS/MS method was successfully established and applied to a pharmacokinetic study in healthy Chinese volunteers after a single oral administration of ibuprofen granules.

Confirmation of Gelsemium Poisoning by Analysis of Koumine, Gelsemine and Gelsenicine in Hair Using LC-MS/MS

A sensitive, accurate, simple, and rapid analytical LC–MS/MS method was developed for the identification and quantification of koumine, gelsemine, and gelsenicine in human hair. Approximately 10 mg of hair was extracted with methanol by cryogenic grinding. The limits of detection (LODs) ranged from 1 to 5 pg/mg, and the limits of quantitation (LOQs) ranged from 2 to 10 pg/mg. The method was linear over a concentration range from the LOQs to 10000 pg/mg, with the linear correlation (R2) of the calibration curves for all analytes above 0.998. The bias varied from -3.6 to 8.8%, while the intra- and inter-day precision Relative Standard Deviation (RSD) values ranged from 1.8–10.8% and 1.9–9.6%, respectively. Recoveries were within the range of 77.8% to 83.3%, and matrix effects were in the range of 74.3% to 87.4%. The present method was suitable for quantitative determination of koumine, gelsemine, and gelsenicine in human hair from one Gelsemium elegans poisoning case. The highest concentrations of koumine, gelsemine, and gelsenicine in the hair segment corresponding to the period of ingestion were 29.4, 17.6, and 4.7 pg/mg, respectively. To our knowledge, this study is the first to describe hair analysis in a Gelsemium poisoning case and to provide quantitative toxicological findings.

Stand out from matrix: Ultra-sensitive LC−MS/MS method for determination of histamine in complex biological samples using derivatization and solid phase extraction

The determination of low abundant endogenous components is a challenge for the clinical samples. Histamine, a crucial endogenous component, fulfils various regulatory and mediatory functions in human, and the change of content is a critical index for the diagnosis of some diseases, especially allergy, asthma, and anaphylactic shock. However, it is challenging to detect histamine because of the low stability and concentration in complex biological samples. Here we developed an ultra-sensitive and accurate LC−MS/MS quantification method based on derivatization, isotope dilution, and solid phase extraction. The derivatization of histamine with diisopropyl phosphite (DIPP) not only enhanced the retention on the LC column but also improved the ionization efficiency. Next, solid phase extraction was applied to remove the interference, which finally resulted in standing out of the trace histamine from the high contents of the matrix. The lowest limit of quantification (LLOQ) was 0.1 pg/mL that is enough low to determine the histamine in one cell and low nano-liter of serum. This approach was successfully applied for the quantification of histamine in clinical serum samples of asthma patients and mast cell treated with chemicals modulating histamine release.

Development and Validation of Stability-Indicating HPLC Method for the Quantification of Levetiracetam in Bulk and Oral Solution: Application to Chemical Kinetics

In the present study, a simple, accurate, precise and specific stability-indicating LC method was developed and validated for the quantification of levetiracetam (LEV) in bulk and oral solution. LEV and its degradation products were separated and resolved successfully on C18 column using a methanol:water (30:70 %, v/v) mixture in the isocratic mode at a flow rate of 1 mL/min. All eluents were detected at 205 nm. In forced degradation experiments, LEV was found to degrade significantly in acid, alkali, wet heat, peroxide mediated oxidation, and photolytic conditions, and found stable under dry heat conditions. Validation experiments showed acceptable accuracy, precision, and specificity of the developed method. Assay of the oral solution was in good agreement with the amount of LEV as per the label claim. The method was applied to investigate chemical kinetics under acid and alkali hydrolysis and the pH rate profile of LEV within a range of pH 2 – 12.

Quantitation of serum pregnanediol-3-glucuronide level in different endocrine statuses by LC–MS/MS

Pregnanediol-3-glucuronide (PdG) is the major terminal metabolite of progesterone, playing an important role in physiological processes, such as the female menstrual cycle, pregnancy (supports gestation), embryogenesis and maternal immune response of humans and other species. Hence, accurate measurement of PdG in serum/plasma is needed for the evaluation of progesterone production. However, such high-specificity determination of PdG is lacking in clinical sample detection. In this study, a highly sensitive and accurate LC–MS/MS method was firstly established for subsequent measurement of PdG in serum of three different female groups: thyroid cancer patients (TCs), healthy controls (HCs) and pregnant women. The factors affecting the sample preparation, MS/MS method, gradient elution program, selection of chromatographic column and internal standard (IS) have been optimized in this study. Compared with enzyme immunoassay (EIA) method, we used LC–MS/MS to shorten analysis time, increase sensitivity, raise specificity, simplify sample preparation, and reduce costs. As a result, the linear range of the method was from 0.38 to 100 ng/mL with a limit of quantification (LOD) of 0.01 ng/mL. Precision assays showed that relative standard deviation (RSD) was less than 10.6, accuracy was between 90.6 % and 110.4 %, and mean recovery was 103.4 %. In addition, the serum PdG/creatinine levels were significantly down-regulated in TCs and up-regulated in pregnant women versus HCs. Receive operating characteristic curve (ROC) analysis enabled to identify TC with a sensitivity of 83.3 %, specificity of 68.0 % and area under curve (AUC) of 0.781 (95 % CI: 0.684 to 0.879), and it enabled to identify pregnant women with a sensitivity of 94.7 %, specificity of 68.5 % and AUC of 0.811 (95 % CI: 0.732 to 0.890). Our results implied that an increase in female serum PdG/creatinine level might be associated with a risk of pregnancy, but serum PdG/creatinine decreasing might be related to a risk of TC.

Simultaneous Determination of 12 Bioactive Constituents in Bupi Yiqi No. 1 Recipe by Ultrahigh-Performance Liquid Chromatography Tandem Mass Spectrometry.

Bupi Yiqi No. 1 Recipe (BPYQ), a traditional Chinese medicine formula, has been widely used as a treatment of multidrug resistance during chemotherapy in colorectal cancer and for the prevention of gastrointestinal cancer recurrence for decades. In this study, a rapid, reliable, and accurate ultrahigh-performance LC (UHPLC) coupled with electrospray ionization (ESI)-tandem MS (MS/MS) method was developed for the simultaneous determination of 12 major bioactive components, including protocatechuic acid, astilbin, rutin, calycosin-7-O-β-D-glucoside, ginsenoside Re, ononin, isoliquiritigenin, calycosin, apigenin, ginsenoside Rb1, formononetin, and glycyrrhizic acid in BPYQ. The chromatographic separation of the analytes was achieved on a Poroshell 120 SB-Aq column (50 × 2.1 mm, 1.7 µm) with a mobile phase of acetonitrile and 0.1% (v/v) formic acid aqueous solution. The flow rate and column temperature were set at 0.4 mL/min and 30°C, respectively. Mass spectrometric detection of the analyses was performed on multiple reaction monitoring mode in positive and negative ESI mode. The established UHPLC-ESI-MS/MS method was validated in terms of the linearity, precision, repeatability, stability, and accuracy. All calibration curves of the 12 compounds showed good linearity, with correlation coefficients (r) greater than 0.9980 within the test ranges. The LODs and LOQs for the 12 compounds were in the ranges of 0.08-1.32 and 0.27-5.28 ng/mL, respectively. The average recoveries of all the standard compounds were between 98.4 and 102.9%, and their relative SD values ranged from 1.24 to 3.78%. The proposed method can provide a meaningful basis for the QC of BPYQ. The established UHPLC-ESI-MS/MS method was demonstrated to be a powerful tool for quantifying the 12 compounds in BPYQ.

Application of in-sample calibration curve methodology for regulated bioanalysis: Critical considerations in method development, validation and sample analysis

Traditionally, for a liquid chromatography tandem mass spectrometry (LC–MS/MS) bioanalytical assay, an external calibration curve is required to achieve accurate quantitation of an analyte. Recently, a novel in-sample calibration curves (ISCC) methodology that can achieve quick and accurate LC–MS/MS bioanalysis without the use of an external calibration curve was reported. The ISCC methodology utilizes the presence of multiple naturally occurring isotopologues of a stable isotopically labeled analyte to construct an in-sample calibration curve for the quantification. This methodology has great potential in many applications, for example biomarker measurement, quantitative proteomics and clinical diagnosis.Here, we assessed the feasibility of applying this ISCC–LC–MS/MS methodology in regulated bioanalysis using BMS-984478, a drug candidate, as the model compound. We also proposed method validation procedures/processes for this new approach for industry peers’ consideration and feedback. A LC–MS/MS method using the ISCC strategy was successfully developed and validated for the quantitative analysis of BMS-984478 in human plasma over the range of 1.33–993.42 ng/mL. The validated ISCC-LC–MS/MS method was compared with a previously validated method using the conventional external calibration curve approach, and the two methods showed equivalent performance. Critical considerations and practical approaches in method development, validation and sample analysis were also discussed. Our work demonstrated that the ISCC–LC–MS/MS methodology is a promising approach for regulated LC–MS/MS bioanalysis. ISCC–LC–MS/MS methodology has its unique advantages and has great potential to be widely applied for various quantitative applications, and may even change the landscape of quantitative analysis.

Development and Validation of LC–MS Method for the Estimation of N-Acetyl-Tryptophan and its Impurities Under Stress Conditions

A highly sensitive and simple high-performance liquid chromatographic–tandem mass spectrometric (LC–MS–MS) assay was developed and validated for the quantification of N-acetyl tryptophan (NAT) in formulations and to identify impurities under different stress conditions. N-acetyl tryptophan was analyzed using a reversed-phase gradient elution after treatment under acidic, basic, oxidative, hydrolytic and thermal stress conditions. Linearity in the calibration curve was obtained at a concentration range of 10–100 µgmL−1 (R2 = 0.9916). The lower limits of detection and quantification were 3.53 and 10.69 µgmL−1. The degradation of NAT was observed maximum under oxidation stress (52.84%) and minimum under thermal stress (10.22%). Three major degradation products were formed under acidic and basic stress conditions, of which tryptophan was the major one. Thermal stress yielded a single major impurity at m/z 230. Water hydrolysis could form dihydroxy-N acetyl tryptophan at m/z 279. Oxidation stress led to the formation of seven major degradation products. The most effective stress condition was found to be oxidative which leads to 52.84% degradation of the drug followed by acidic stress (34.64%) and basic stress (15.66%). The present study showed an accurate, precise and sensitive LC–MS–MS method for the systematic investigation of NAT and its impurities in formulations.

Simultaneous determination of multiple compounds of Da-Huang-Xiao-Shi decoction in rat plasma by LC-MS/MS and its application in a pharmacokinetic study

Da-Huang-Xiao-Shi decoction (DHXSD), a traditional Chinese medicinal formula, has been used mainly to treat jaundice for more than 1700 years in China. In this study, we developed a rapid, sensitive, and accurate LC–MS/MS method to simultaneously determine multiple, potentially bioactive compounds of DHXSD, including five alkaloids (berberine, phellodendrine, palmatine, jatrorrhizine, and magnoflorine), five anthraquinones (rhein, aloe-emodin, emodin, chrysophanol, and physcion), two iridoid glycosides (geniposide and genipin 1-gentiobioside), and one iridoid aglycone (genipin) in rat plasma. Plasma samples collected from rats were treated immediately with 5% acetic acid to avoid the degradation of genipin. After protein precipitation with acetonitrile containing 5% acetic acid, the compounds were reconstituted in acetonitrile–water (50:50, v/v) solution containing 6.5% formic acid and separated on the ACQUITY™ UPLC BEH C18 column (2.1 × 100 mm; 1.7 μm) using a mobile phase composed of 2 mM ammonium formate in water (solvent A) and acetonitrile (solvent B) at a flow rate of 0.3 mL/min. Quantitation was performed on a Triple Quand 5500 tandem mass spectrometer coupled with an electrospray ionization (ESI) source. Multiple reaction monitoring (MRM) was used to quantify compounds in positive and negative ion modes. The method validation results showed that the specificity, linearity, precision and accuracy, recovery, matrix effect, and stability of the 13 compounds met the requirements for their quantitation in biological samples. This newly established method was successfully used in a pharmacokinetic study on rats orally treated with DHXSD. Besides, glucuronide and sulfate metabolites were also determined in rat plasma after hydrolysis. This is the first method developed for the simultaneous quantification of multiple compounds of DHXSD in vivo. Our study provides relevant information on the pharmacokinetics of DHXSD and the relationship between the compounds of DHXSD and their therapeutic effects.

Reductive Desulfuration as an Important Tool in Detection of Small Molecule Modifications to Payload of Antibody Drug Conjugates.

A high resolution accurate mass LC-MS method was developed to facilitate the characterization of a subset of antibody drug conjugate (ADC) biotherapeutics, where the payload is linked to the antibody by a thioether bond. Desulfuration of the thioether linker was optimized for release of the payload to take advantage of the high resolution and high mass accuracy of the Orbitrap to characterize metabolism of the payload. Two model ADCs, trastuzumab emtansine (T-DM1) and SigmaMAb dansyl-cadavarine-SMCC (SigmaMAb ADC mimic) were selected for optimization of the desulfuration reaction as a function of reaction time, pH, organic solvent, and chaotropic reagents (urea, guanidine HCl) by monitoring the yield of released desulfurated DM1 from T-DM1 and desulfurated dansyl-cadaverine-SMCC from SigmaMAb ADC mimic, respectively. The optimized desulfuration technique was successfully applied to enable characterization of the ADC following its incubation in hepatocytes, liver microsomes, and buffers, as illustrated by the identification of a hydrolyzed thiosuccinimide ring of SigmaMAb ADC mimic following incubation in buffer for 48 h. The results from this study demonstrate that the chemical cleavage of thioether bond by desulfuration is simple, efficient, and specific. This technique is useful in characterization of metabolism on the payload of ADC to provide guidance for improvement of its biopharmaceutical profile. This is the first report on characterization of modification to payload of ADC following desulfuration.

Determination of Vitamin D3 Content in High, Low and Zero Fat Food Using High Performance Liquid Chromatography

AbstractBackground: Vitamin D, has a significant role in bone metabolism and helps calcium absorption in the body. There are only few vitamin D assay methods available for zero fat food.Aims of Study: (1) To develop an accurate and sensitive LC method for the quantification of vitamin D3 in food by optimization of each step of the analytical method: Extraction, sample preparation, separation and detection. (2) To validate the developed method. (3) To apply the method to quantify the total vitamin D3 in food from several species.Material and Methods: In this study, a rapid, simple, and economical reversed phase liquid chromatographic method was described for the determination of vitamin D3, in some high, low and zero fat samples (milk products, cereal and chewing gum samples). The isolation of fat soluble vitamins includes a saponification step and an extraction step with petroleum ether and diethyl ether. The vitamin D3 content of samples was determined by reversed phase liquid chromatog-raphy. Ultra violet-Visible (UV-VIS) detector and C18 column were used for this purpose.Results: The linearity of standard curves of vitamin D3 were 10-200 mg/ml expressed of the correlation coefficient r2 = 0.9992). The detection (LOD) and quantification (LOQ) limits were 5.09 and 15.42mg/ml, respectively. The accuracy was 101.37±4.37.Conclusion: The described reversed-phase HPLC method is favorable compared with other published HPLC-UV methods (20 and 21) because of its stability-indicating nature, short run time and wide analytical range with outstanding linearity, accuracy and precision. The proposed method allows the determination of vitamin D3 in a single chromatographic run and is suitable for the analysis of the stability of vitamin D3. The obtained results from the assay of vitamin D3 in commer-cial nutrition supplements confirmed that the method is appropriate for the routine analysis of various food samples.