Microfluidic Lab-on-CMOS Packaging Using Wafer-Level Molding and 3D-Printed Interconnects.

Lab-on-a-chip (LoC) technologies continue to promise lower cost and more accessible platforms for performing biomedical testing in low-cost and disposable form factors. Lab-on-CMOS or lab-on-microchip methods extend this paradigm by merging passive LoC systems with active complementary metal-oxide semiconductor (CMOS) integrated circuits (IC) to enable front-end signal conditioning and digitization immediately next to sensors in fluid channels. However, integrating ICs with microfluidics remains a challenge due to size mismatch and geometric constraints, such as non-planar wirebonds or flip-chip approaches in conflict with planar microfluidics. In this work, we present a hybrid packaging solution for IC-enabled microfluidic sensor systems. Our approach uses a combination of wafer-level molding and direct-write 3D printed interconnects, which are compatible with post-fabrication of planar dielectric and microfluidic layers. In addition, high-resolution direct-write printing can be used to rapidly fabricate electrical interconnects at a scale compatible with IC packaging without the need for fixed tooling. Two demonstration sensor-in-package systems with integrated microfluidics are shown, including measurement of electrical impedance and optical scattering to detect and size particles flowing through microfluidic channels over or adjacent to CMOS sensor and read-out ICs. The approach enables fabrication of impedance measurement electrodes less than 1 mm from the readout IC, directly on package surface. As shown, direct fluid contact with the IC surface is prevented by passivation, but long-term this approach can also enable fluid access to IC-integrated electrodes or other top-level IC features, making it broadly enabling for lab-on-CMOS applications.

A 10 M Ω, 50 kHz-40 MHz Impedance Measurement Architecture for Source-Differential Flow Cytometry.

A low-power, impedance-based integrated circuit (IC) readout architecture is presented for cell analysis and cytometry applications. A three-electrode layout and source-differential excitation cancels baseline current prior to the sensor front-end, which enables the use of a high-gain readout circuit for the difference current. A lock-in architecture is employed with down-conversion and up-conversion in the feedback loop, enabling high closed-loop gain (up to 10 M Ω) and high bandwidth (up to 40 MHz). A hybrid-RC feedback network mitigates the SNR degradation seen over a wide operating frequency range when using purely capacitive feedback. The effect of phase shift on the closed-loop system gain and noise performance are analyzed in detail, along with optimization strategies, and the design includes fine-grained phase adjustment to minimize phase error. The impedance sensor was fabricated in a 0.18 µ m CMOS process and consumes 9.7 mW with an operating frequency from 50 kHz to 40 MHz and provides adjustable bandwidth. Measurements demonstrate that the impedance sensor achieves 6 pA [Formula: see text] input-referred noise over 200 Hz bandwidth at 0.5 MHz modulation frequency. Combined with a microfluidic flow cell, measured results using this source-differential measurement approach are presented using both monodisperse and polydisperse sample solutions and demonstrate single-cell resolution, detecting 3 µ m diameter particles in solution with 22 dB SNR.

A Batteryless Motion-Adaptive Heartbeat Detection System-on-Chip Powered by Human Body Heat.

This paper presents a batteryless heartbeat detection system-on-chip (SoC) powered by human body heat. An adaptive threshold generation architecture using pulse-width locked loop (PWLL) is developed to detect heartbeats from electrocardiogram (ECG) in the presence of motion artifacts. The sensing system is autonomously powered by harvesting thermal energy from human body heat using a thermoelectric generator (TEG) coupled to a low-voltage, self-starting boost converter and integrated power management system. The SoC was implemented in a 0.18 µm CMOS process and is fully functional with a minimum input power of 20 µW, provided by a portable TEG at 20 mV with a ~0.5 °C temperature gradient. The complete system demonstrates motion-adaptive, power-autonomous heartbeat detection for sustainable healthcare using wearable devices.

DC-100 kHz Tunable Readout IC for Impedance Spectroscopy and Amperometric Measurement of Electrochemical Sensors.

This paper presents a low-noise, front-end sensor IC that includes both AC impedance spectroscopy and DC amperometric measurement capabilities for electrochemical and biosensor applications. A common-gate current buffer topology is proposed that supports both current-mode and voltage-mode sensor signals to allow an input frequency range from DC to 100 kHz. Low-noise operation is achieved across a wide input frequency range using tunable high-pass and low-pass frequency response. In addition, an incremental delta-sigma modulator with embedded frequency response analysis serves as both on-chip impedance analyzer and current-driven analog-to-digital converter. Implemented using a 0.18 µm CMOS process, this work achieves 45 fA/√Hz input current noise density at 1 kHz. Input dynamic range exceeding 80 dB is achieved up to 10 kHz bandwidth, with a maximum of 104 dB dynamic range at 10 Hz.

Insights into the Authentic Active Ingredients and Action Sites of Oral Exogenous Glutathione in the Treatment of Ischemic Brain Injury Based on Pharmacokinetic-Pharmacodynamic Studies.

Glutathione (GSH) has been reported to be closely related to various diseases of the central nervous system, yet its authentic active ingredients and action sites remain unclear. In the present study, oral exogenous GSH significantly alleviated ischemic brain injury, but this result was inconsistent with its low bioavailability and blood-brain barrier (BBB) permeability. To ascertain the exposure of GSH-derived ingredients, including GSH, cysteine (CYS), glutamate (Glu), glycine (GLY), CYS-GLY, and γ-glutamylcysteine (γ-GC) were systematically studied both in vitro and in vivo. The outcomes demonstrated that oral GSH not only increases the GSH and CYS levels in rat striatum and cortex, but it also can decrease the rise of intracerebral Glu concentration caused by ischemia/reperfusion surgery. Then the influence of GSH on the BBB was investigated via measuring IgG leakage, intracerebral endotoxin, and tight-junction proteins. All indicators showed that GSH dosing can repair the destroyed BBB. Oral GSH greatly enhances the exposure of GSH, CYS, CYS-GLY, and γ-GC in rat duodenum, jejunum, ileum, and colon. Accumulating evidence reveals a close link between brain injury and gastrointestinal dysfunction. Our findings further suggest that oral GSH significantly improves intestinal inflammatory damage and barrier disruptions. In conclusion, oral GSH can have a direct therapeutic role in brain injury by stabilizing intracerebral levels of GSH, CYS, and Glu. It can also play an indirect therapeutic role by enhancing the intestinal exposure of GSH, CYS, CYS-GLY, and γ-GC and improving intestinal barrier disruptions. SIGNIFICANCE STATEMENT: The authentic active ingredients and action sites of exogenous glutathione (GSH) in the treatment of ischemic brain injury are unclear. We have shown that oral exogenous GSH not only stabilizes intracerebral levels of GSH, cysteine (CYS), and glutamate (Glu) to act directly on brain injury, but it can also exert an indirect therapeutic role by improving intestinal barrier disruptions. These findings have great significance for revealing the therapeutic effect of GSH on ischemic brain injury and for promoting its further development and clinical application.

Systematically identifying the hepatoprotective ingredients of schisandra lignan extract from pharmacokinetic and pharmacodynamic perspectives.

BACKGROUND: Herbal medicines (HMs) have been proven to be productive sources of leads for the development of drugs. To date approximately 150 lignans have been identified from Schisandra sphenanthera. Hepatoprotective activity is a well-known characteristic of schisandra lignans, yet the authentic types of active lignans are still not well known. PURPOSE: The present study aimed to develop a reliable and efficient strategy for identifying the hepatoprotective ingredients of schisandra lignan extract (SLE). METHODS: SLEs were prepared by extracting Schisandra sphenanthera powder using 10%, 50% and 90% ethanol (w/w 1:10) combining 5-fold volume of ethyl acetate. The schisandra lignans in SLEs were qualitatively analyzed based on liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LCMS-IT-TOF). Preparative liquid chromatography (PLC) was used to collect ingredient fractions. The hepatoprotective activity of schisandra lignans was systematically investigated on in vivo and in vitro models. RESULTS: The SLE extracted by 50% ethanol and 5-fold volume of ethyl acetate (50%SLE) had the highest lignan content and exhibited significantly stronger hepatoprotective activity than other SLEs (P <  0.01). The hepatoprotective effect of 50%SLE mainly attributed to the SLE segment which collected from 12 to 22 min by PLC. Schisantherin A (Sth A) was confirmed as the most promising hepatoprotective drug in Schisandra sphenanthera due to high content in crude materials, high exposure level in vivo and high efficiency on APAP-induced hepatotoxicity. CONCLUSION: The hepatoprotective ingredients of SLEs were systematically investigated based on the presently developed approach, and Sth A was identified as the optimum hepatoprotective candidate in Schisandra sphenanthera.

Is the combinational administration of doxorubicin and glutathione a reasonable proposal?

The combinational administration of antioxidants and chemotherapeutic agents during conventional cancer treatment is among one of the most controversial areas in oncology. Although the data on the combinational usage of doxorubicin (DOX) and glutathione (GSH) agents have been explored for over 20 years, the duration, administration route, and authentic rationality have not yet been fully understood yet. In the current study, we systematically investigated the pharmacokinetics (PK) and pharmacodynamics (PD) with both in vivo and in vitro models to elucidate the influence of GSH on the toxicity and efficacy of DOX. We first studied the cardioprotective and hepatoprotective effects of GSH in Balb/c mice, H9c2, and HL7702 cells. We showed that coadministration of exogenous GSH (5, 50, and 500 mg/kg per day, intragastric) significantly attenuated DOX-induced cardiotoxicity and hepatotoxicity by increasing intracellular GSH levels, whereas the elevated GSH concentrations did not affect the exposure of DOX in mouse heart and liver. From PK and PD perspectives, then the influences of GSH on the chemotherapeutic efficacy of DOX were investigated in xenografted nude mice and cancer cell models, including MCF-7, HepG2, and Caco-2 cells, which revealed that administration of exogenous GSH dose-dependently attenuated the anticancer efficacy of DOX in vivo and in vitro, although the elevated GSH levels neither influenced the concentration of DOX in tumors in vivo, nor the uptake of DOX in MCF-7 tumor cells in vitro. Based on the results we suggest that the combined administration of GSH and DOX should be contraindicated during chemotherapy unless DOX has caused serious hepatotoxicity and cardiotoxicity.

Activated charcoal significantly improved the reliability of methods for quantitative analysis of endogenous substances in biological specimens: Glutathione and cysteine as cases.

When developing a quantitative assay for exogenous or endogenous compounds, guidelines for method validation normally recommend that the biological specimens should be prepared in corresponding authentic matrices, yet "analyte-free authentic matrices" is in general not available. It is generally known that GSH and CYS are endogenous compounds and present in both prokaryotes and eukaryotes. Herein, an efficient approach for the quantitative analysis of endogenous substances in biological specimens was developed, and glutathione (GSH) & cysteine (CYS) were chosen as model endogenous substances. Activated carbon (AC), a common adsorbent for the adsorption of environmental pollutants, was used to remove the endogenous GSH and CYS and prepare "GSH&CYS-free biological matrix". The endogenous GSH and CYS in mouse plasma, blood and liver homogenate were found can be almost removed via incubating with 100 mg of AC for 2 h. After optimizing the derivatization reagents, internal standard and analytical parameters, a reliable quantitative assay of GSH and CYS in mouse plasma, blood and liver homogenate was developed and validated on LC-ESI-MS/MS using corresponding AC-adsorbed mouse biological matrices. The validation results indicated that the developed method provided suitable accuracy, sensitivity, specificity and high throughput for the analysis of GSH and CYS. Finally, the developed LC-ESI-MS/MS assay was successfully applied to measure the concentrations of GSH and CYS in liver injury mice. The presently developed methodology could be widely applied in the quantitative analysis of endogenous compounds in various complex mixtures such as biological, herbal and environmental samples.

Pharmacokinetic and pharmacodynamic evidence for developing an oral formulation of octreotide against gastric mucosal injury.

Among the somatostatin analogues, octreotide (OCT) is the most commonly used in clinic via intravenous or subcutaneous injection to treat various diseases caused by increased secretion of growth hormone, gastrin or insulin. In order to assesse the feasibility of developing oral formulations of OCT, we conducted systematical pharmacokinetic and pharmacodynamic analyses of OCT in several animal models. The pharmacokinetic studies in rats showed that intragastric administration of OCT had extremely low bioavailability (<0.5%), but it could specifically distribute to the gastric mucosa due to the high expression of somatostatin receptor 2 (SSTR2) in the rat stomach. The pharmacodynamic studies revealed that intragastric administration of OCT dose-dependently protected against gastric mucosal injury (GMI) in mice with WIRS-induced mouse gastric ulcers, which were comparable to those achieved by intravenous injection of OCT, and this effect was markedly attenuated by co-administration of CYN-154806, an antagonist of SSTR2. In pyloric ligation-induced ulcer mice, we further demonstrated that OCT significantly reduced the secretion of gastric acid via down-regulating the level of gastrin, which was responsible for the protective effect of OCT against GMI. Overall, we have provided pharmacokinetic and pharmacodynamic evidence for the feasibility of developing an oral formulation of OCT. Most importantly, the influence of SSTR2 on the pharmacokinetics and pharmacodynamics of OCT suggested that an oral formulation of OCT might be applicable for other clinical indications, including neuroendocrine neoplasms and pituitary adenoma due to the overexpression of SSTR2 on these tumor cells.

Low Cerebral Exposure Cannot Hinder the Neuroprotective Effects of Panax Notoginsenosides.

A bidirectional route of communication between the gastrointestinal tract and the central nervous system, termed the "gut-brain axis," is becoming increasingly relevant to treatment of cerebral damage. Panax Notoginsenoside extract (PNE) is popular for prevention and treatment of cardio-cerebrovascular ischemic diseases although plasma and cerebral exposure levels are extremely low. To date, the mechanisms underlying the neuroprotective effects of PNE remain largely unknown. In the present study, the neuroprotective effects of PNE were systematically studied via investigation of the regulation by PNE of the gastrointestinal microbial community and γ aminobutyric acid (GABA) receptors. The results demonstrated that pretreatment with PNE exerted a remarkable neuroprotective effect on focal cerebral ischemia/reperfusion (I/R) injury in rats, and the efficiency was attenuated in germ-free rats. Pretreatment with PNE could significantly prevent downregulation of Bifidobacterium longum (B.L) caused by I/R surgery, and colonization by B.L could also exert neuroprotective effects. More importantly, both PNE and B.L could upregulate the expression of GABA receptors in the hippocampus of I/R rats, and coadministration of a GABA-B receptor antagonist could significantly attenuate the neuroprotective effects of PNE and B.L. The study above suggests that the neuroprotective effects of PNE may be largely attributable to its regulation of intestinal flora, and oral treatment with B.L was also useful in therapy of ischemia/reperfusion injury (I/R) by upregulating GABA-B receptors.

An integrated strategy for the quantitative analysis of endogenous proteins: A case of gender-dependent expression of P450 enzymes in rat liver microsome.

Liquid chromatography mass spectrometry based methods provide powerful tools for protein analysis. Cytochromes P450 (CYPs), the most important drug metabolic enzymes, always exhibit sex-dependent expression patterns and metabolic activities. To date, analysis of CYPs based on mass spectrometry is still facing critical technical challenges due to the complexity and diversity of CYP isoforms besides lack of corresponding standards. The aim of present work consisted in developing a label-free qualitative and quantitative strategy for endogenous proteins, and then applying to the gender-difference study for CYPs in rat liver microsomes (RLMs). Initially, trypsin digested RLM specimens were analyzed by the nanoLC-LTQ-Orbitrap MS/MS. Skyline, an open source and freely available software for targeted proteomics research, was then used to screen the main CYP isoforms in RLMs under a series of criteria automatically, and a total of 40 and 39 CYP isoforms were identified in male and female RLMs, respectively. More importantly, a robust quantitative method in a tandem mass spectrometry-multiple reaction mode (MS/MS-MRM) was built and optimized under the help of Skyline, and successfully applied into the CYP gender difference study in RLMs. In this process, a simple and accurate approach named 'Standard Curve Slope" (SCS) was established based on the difference of standard curve slopes of CYPs between female and male RLMs in order to assess the gender difference of CYPs in RLMs. This presently developed methodology and approach could be widely used in the protein regulation study during drug pharmacological mechanism research.

Optimization and evaluation of MALDI TOF mass spectrometric imaging for quantification of orally dosed octreotide in mouse tissues.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF-MSI) has received considerable attention in recent years since it allows molecular mapping of diverse bimolecular in animal/plant tissue sections, although some barriers still exist in absolute pixel-to-pixel quantification. Octreotide, a synthetic somatostatin analogue, has been widely used to prevent gastrointestine bleeding in the clinic. The aim of the present study is to develop a MALDI-TOF-MSI method for quantitatively visualizing spatial distribution of octreotide in mouse tissues. In this process, a structurally similar internal standard was spotted onto tissue section together with matrix solution to minimize signal variation and give excellent quantitative results. The 2,5-dihydroxybenzoic acid was chosen as the most suitable matrix via comparing the signal/noise generated by MALDI-TOF-MSI after cocrystallization of octreotide with different matrix candidates. The reliability of MALDI-TOF-MSI, with respect to linearity, sensitivity and precision, was tested via measuring octreotide in fresh tissue slices at different concentrations. The validated method was then successfully applied to visualize the distribution of octreotide in mouse tissues after oral administration of octreotide at 20mg/kg. The results demonstrated that MALDI-TOF-MSI could not only clearly visualize the spatial distribution of octreotide, but also make the calculation of the key pharmacokinetic parameters (Tmax and t1/2) possible. More importantly, the tissue concentration-time curves of octreotide determined by MALDI-TOF-MSI agreed well with those measured based on LC-MS/MS.These findings illustrate the potential of MALDI-TOF-MSI in pharmacokinetic profiling during drug development.

Pharmacokinetic study based on a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight imaging mass microscope combined with a novel relative exposure approach: A case of octreotide in mouse target tissues.

Application of imaging mass spectrometry in drug pharmacokinetics remains challenging due to its weak quantitative capability. Herein, an imaging mass microscope (iMScope), equipped with an optical microscope, an atmospheric pressure ion-source chamber for matrix-assisted laser desorption/ionization (AP-MALDI) and a hybrid quadrupole ion trap time-of-flight (QIT-TOF) analyzer, was first validated and applied to visualize drug disposition in vivo. The distribution and elimination rate of the therapeutic peptide octreotide, a long-acting analogue of the natural hormone somatostatin, was first calculated based on the data determined by iMScope system combining a novel relative exposure strategy. Microspotted pixel-to-pixel quantitative iMScope provided a relative amount of octreotide presented in a thin stomach/intestinal section while preserving its original spatial distribution. The images of dosed mouse stomach clearly demonstrated the transport process of octreotide from the mucosal layer to the muscle side. More importantly, octreotide was found to eliminate from the intestines rapidly, the absorption peak time (Tmax) appeared at 40 min and the half-life time (t1/2) was calculated as 37.7 min according to the elimination curves. Comparisons to the LC-MS/MS data adequately indicated that the quantification approach and methodology based on the iMScope was reliable and practicable for drug pharmacokinetic study.

Bioanalytical assay development and validation for simultaneous quantification of five schisandra lignans in rat primary hepatocytes based on LC-MS/MS: application to a real-time uptake study for Schisandra Lignan Extract.

Schisandra lignans, mainly including schizandrol A, schizandrol B, schisantherin A, schizandrin A, schizandrin B, etc., are the major active ingredients of Schisandra chinensis. In the present study, a robust liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous quantification of schisandra lignans in rat primary hepatocytes. Lovastatin was used as an internal standard, and chromatographic separation was achieved on a Shimadzu C18 column with a gradient elution at the flow rate of 0.2 mL/min. All of the analytes were detected in multiple reaction monitoring mode with positive electrospray ionization since the sodium adduct ion [M + Na]+ was observed as the most intensive peak in the MS spectrum. For schizandrol A, schisantherin A and schizandrin A, the dynamic range was within 2-1000 ng/mg protein, and the linear range of schizandrol B and schizandrin B was from 5 to 1000 ng/mg protein. The intra- and inter-day precision was <15% and the accuracy (relative error) ranged from -15 to 15%. No significant variation was observed in the stability tests. The validated method was then successfully applied to the time-dependent uptake study for the Schisandra Lignan Extract in rat primary hepatocytes.

Qualitatively and quantitatively investigating the regulation of intestinal microbiota on the metabolism of panax notoginseng saponins.

ETHNOPHARMACOLOGICAL RELEVANCE: Intestinal microflora plays crucial roles in modulating pharmacokinetic characteristics and pharmacological actions of active ingredients in traditional Chinese medicines (TCMs). However, the exact impact of altered intestinal microflora affecting the biotransformation of TCMs remains poorly understood. AIMS OF THE STUDY: This study aimed to reveal the specific enterobacteria which dominate the metabolism of panax notoginseng saponins (PNSs) via exploring the relationship between bacterial community structures and the metabolic profiles of PNSs. MATERIALS AND METHODS: 2, 4, 6-Trinitrobenzenesulphonic acid (TNBS)-challenged and pseudo germ-free (pseudo GF) rats, which prepared by treating TNBS and antibiotic cocktail, respectively, were employed to investigate the influence of intestinal microflora on the PNS metabolic profiles. Firstly, the bacterial community structures of the conventional, TNBS-challenged and pseudo GF rat intestinal microflora were compared via 16S rDNA amplicon sequencing technique. Then, the biotransformation of protopanaxadiol-type PNSs (ginsenoside Rb1, Rb2 and Rd), protopanaxatriol-type PNSs (ginsenoside Re, Rf, Rg1 and notoginsenoside R1) and Panax notoginseng extract (PNE) in conventional, TNBS-challenged and pseudo GF rat intestinal microbiota was systematically studied from qualitative and quantitative angles based on LC-triple-TOF/MS system. Besides, glycosidases (ß-glucosidase and ß-xylosidase), predominant enzymes responsible for the deglycosylation of PNSs, were measured by the glycosidases assay kits. RESULTS: Significant differences in the bacterial community structure on phylum, class, order, family, and genera levels were observed among the conventional, TNBS-challenged and pseudo GF rats. Most of the metabolites in TNBS-challenged rat intestinal microflora were identified as the deglycosylation products, and had slightly lower exposure levels than those in the conventional rats. In the pseudo GF group, the peak area of metabolites formed by loss of glucose, xylose and rhamnose was significantly lower than that in the conventional group. Importantly, the exposure levels of the deglycosylated metabolites were found have a high correlation with the alteration of glycosidase activities and proteobacteria population. Several other metabolites, which formed by oxidation, dehydrogenation, demethylation, etc, had higher relative exposure in pseudo GF group, which implicated that the up-regulation of Bacteroidetes could enhance the activities of some redox enzymes in intestinal microbiota. CONCLUSION: The metabolism of PNSs was greatly influenced by intestinal microflora. Proteobacteria may affect the deglycosylated metabolism of PNSs via regulating the activities of glycosidases. Besides, up-regulation of Bacteroidetes was likely to promote the redox metabolism of PNSs via improving the activities of redox metabolic enzymes in intestinal microflora.

Development of a novel sectional multiple filtering scheme for rapid screening and classifying metabolites of ziyuglycoside II in rat liver and excreta specimen based on high-resolution mass spectrometry.

Ziyuglycoside II, one of the major effective ingredients of Sanguisorba officinalis L., had various pharmacological activities including anticancer, anti-inflammation and anti-oxidation, etc. Better understanding of the pharmacology and toxicology of ziyuglycoside II requires the detailed elucidation of its biologic fates in vivo. Herein, the metabolic fate of ziyuglycoside II in rats was investigated based on liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS). To accelerate and simplify the process of metabolite identification from complicated biological matrix, the sectional multiple filtering (SMF) scheme was designed according to the relationship among the molecular weight (MW), mass defect (MD) and retention time (tR) of the metabolites. SMF-I (MW: 700-850Da, MD: 0.40-0.45Da, tR: 4.0-10.0min), SMF-II (MW: 550-700Da, MD: 0.30-0.40Da, tR: 6.0-14.0min) and SMF-III (MW: 400-550Da, MD at 0.25-0.35Da, tR at 9.5-16.0min) were built and utilized to screen phase II conjugations and phase I redox metabolites and deglycosylated derivatives, respectively. As a result, dozens of metabolites, including glucuronic conjugates, hydroxylation, oxidization, dehydration and deglycosylation products, were rapidly discovered, classified and structural identified in rat urine and feces based on SMF scheme and accurate MS(1)/MS(2) information. Obviously, the SMF technique showed superior efficiency and selectivity in ziyuglycoside II metabolite identification. More importantly, SMF would find its extensive application in, but not limited to, the metabolic study for single drug or homologous compounds in traditional Chinese medicine.

The metabolic and pharmacokinetic studies for HM-3 in rats based on LC-Q-TOF/MS and LC-MS/MS combing a convenient biological sample processing method.

In this contribution, the metabolic and pharmacokinetic characteristics for the therapeutic peptide HM-3 were investigated using LC-Q-TOF/MS and LC-MS/MS systems combing a fast biological sample processing method. According to the accurate MS(1) and MS(2) data generated by LC-Q-TOF/MS, a total of 6 metabolites in rats were detected and tentatively identified as the degradation products which formed by successive loss of amino acid from HM-3. The structures of the 2 main metabolites (M1 and M2) were confirmed by comparing the chromatographic and mass spectrographic characteristics with the corresponding synthetic standards. Then, an absolute quantitative analysis method based on LC-MS/MS system was built and fully validated with respect to linearity, sensitivity, accuracy, precision, matrix effect, stability, etc. The results indicated that HM-3, M1 and M2 were linear in peak area ratios over the concentration range of 0.5-200.0ng/mL with a correlation coefficient>0.99. The intra-day and inter-day precisions (RSD%) were less than 15%, and the accuracy was below 10% in terms of RE%. The validated method was then successfully applied to the studies of preclinical pharmacokinetics for HM-3.

A robust LC-MS/MS method for the determination of pidotimod in different biological matrixes and its application to in vivo and in vitro pharmacokinetic studies.

Pidotimod, (R)-3-[(S)-(5-oxo-2-pyrrolidinyl) carbonyl]-thiazolidine-4-carboxylic acid, was frequently used to treat children with recurrent respiratory infections. Preclinical pharmacokinetics of pidotimod was still rarely reported to date. Herein, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine pidotimod in rat plasma, tissue homogenate and Caco-2 cells. In this process, phenacetin was chosen as the internal standard due to its similarity in chromatographic and mass spectrographic characteristics with pidotimod. The plasma calibration curves were established within the concentration range of 0.01-10.00µg/mL, and similar linear curves were built using tissue homogenate and Caco-2 cells. The calibration curves for all biological samples showed good linearity (r>0.99) over the concentration ranges tested. The intra- and inter-day precision (RSD, %) values were below 15% and accuracy (RE, %) was ranged from -15% to 15% at all quality control levels. For plasma, tissue homogenate and Caco-2 cells, no obvious matrix effect was found, and the average recoveries were all above 75%. Thus, the method demonstrated excellent accuracy, precision and robustness for high throughput applications, and was then successfully applied to the studies of absorption in rat plasma, distribution in rat tissues and intracellular uptake characteristics in Caco-2 cells for pidotimod.