Miniature mass spectrometer-based point-of-care assay for quantification of metformin and sitagliptin in human blood and urine.

Metformin (MET) and sitagliptin (STG) are widely used as the first-line and long-term oral hypoglycemic agents for managing type 2 diabetes mellitus (T2DM). However, the current lack of convenient and rapid measurement methods poses a challenge for individualized management. This study developed a point-of-care (POC) assay method utilizing a miniature mass spectrometer, enabling rapid and accurate quantification of MET and STG concentrations in human blood and urine. By combining the miniature mass spectrometer with paper spray ionization, this method simplifies the process into three to four steps, requires minimal amounts of bodily fluids (50 µL of blood and 2 µL of urine), and is able to obtain quantification results within approximately 2 min. Stable isotope-labeled internal standards were employed to enhance the accuracy and stability of measurement. The MS/MS responses exhibited good linear relationship with concentration, with relative standard deviations (RSDs) below 25%. It has the potential to provide immediate treatment feedback and decision support for patients and healthcare professionals in clinical practice.

A Pharmacokinetic Analysis of Hemodialysis for Metformin-Associated Lactic Acidosis.

OBJECTIVE: Although hemodialysis is recommended for patients with severe metformin-associated lactic acidosis (MALA), the amount of metformin removed by hemodialysis is poorly documented. We analyzed endogenous clearance and hemodialysis clearance in a patient with MALA. METHODS: A 62-year-old man with a history of type II diabetes mellitus presented after several days of vomiting and diarrhea and was found to have acute kidney injury (AKI) and severe acidemia. Initial serum metformin concentration was 315.34 µmol/L (40.73 µg/mL) (typical therapeutic concentrations 1-2 µg/mL). He underwent 6 h of hemodialysis. We collected hourly whole blood, serum, urine, and dialysate metformin concentrations. Blood, urine, and dialysate samples were analyzed, and clearances were determined using standard pharmacokinetic calculations. RESULTS: The total amount of metformin removed by 6 h of hemodialysis was 888 mg, approximately equivalent to one therapeutic dose. Approximately 142 mg of metformin was cleared in the urine during this time. His acid-base status and creatinine improved over the following days. No further hemodialysis was required. CONCLUSION: We report a case of MALA likely secondary to AKI and severe volume depletion. The patient improved with supportive care, sodium bicarbonate, and hemodialysis. Analysis of whole blood, serum, urine, and dialysate concentrations showed limited efficacy of hemodialysis in the removal of metformin from blood, contrary to previously published data. Despite evidence of acute kidney injury, a relatively large amount of metformin was eliminated in the urine while the patient was undergoing hemodialysis. These data suggest that clinical improvement is likely due to factors besides removal of metformin.

The effects of high-fat diet and metformin on urinary metabolites in diabetes and prediabetes rat models.

High-fat diet (HFD) interferes with the dietary plan of patients with type 2 diabetes mellitus (T2DM). However, many diabetes patients consume food with higher fat content for a better taste bud experience. In this study, we examined the effect of HFD on rats at the early onset of diabetes and prediabetes by supplementing their feed with palm olein oil to provide a fat content representing 39% of total calorie intake. Urinary profile generated from liquid chromatography-mass spectrometry analysis was used to construct the orthogonal partial least squares discriminant analysis (OPLS-DA) score plots. The data provide insights into the physiological state of an organism. Healthy rats fed with normal chow (NC) and HFD cannot be distinguished by their urinary metabolite profiles, whereas diabetic and prediabetic rats showed a clear separation in OPLS-DA profile between the two diets, indicating a change in their physiological state. Metformin treatment altered the metabolomics profiles of diabetic rats and lowered their blood sugar levels. For prediabetic rats, metformin treatment on both NC- and HFD-fed rats not only reduced their blood sugar levels to normal but also altered the urinary metabolite profile to be more like healthy rats. The use of metformin is therefore beneficial at the prediabetes stage.

A drug-drug interaction study to evaluate the impact of peficitinib on OCT1- and MATE1-mediated transport of metformin in healthy volunteers.

PURPOSE: Peficitinib is an oral pan-Janus kinase inhibitor for the treatment of rheumatoid arthritis. Co-administration of peficitinib with metformin, a type 2 diabetes therapy, can occur in clinical practice. Hepatic and renal uptake of metformin is mediated by organic cation transporter 1 (OCT1) and OCT2, respectively, and its renal excretion by multidrug and toxin extrusion 1 (MATE1) and MATE2-K. This study investigated the effect of peficitinib on metformin pharmacokinetics in vitro and in healthy volunteers. METHODS: Inhibitory effects of peficitinib and its metabolite H2 on metformin uptake into human OCT1/2- and MATE1/2-K-expressing cells were assessed in vitro. In an open-label, drug-drug interaction study, 24 healthy volunteers received a single dose of metformin 750 mg on Days 1 and 10, and a single dose of peficitinib 150 mg on Days 3 and 5-11. Blood and urine samples were collected pre-dose on Days 1 and 10, and at intervals ≤ 48 h post-dose. Metformin concentration was determined by liquid chromatography-tandem mass spectrometry and its pharmacokinetic parameters calculated. RESULTS: Peficitinib, but not H2, inhibited metformin uptake into OCT1- and MATE1/2-K-expressing cells. Repeated-dose administration of peficitinib reduced metformin area under the concentration-time curve from 0 h extrapolated to infinity (AUCinf) by 17.4%, maximum plasma concentration (Cmax) by 17.0%, and renal clearance (CLR) by 12.9%. Co-administration of peficitinib with metformin was generally well tolerated. CONCLUSION: Slight changes in AUCinf, Cmax and CLR of metformin were observed when co-administered with peficitinib; however, these changes were considered not clinically relevant.

Urinary detection of rapid-acting insulin analogs in healthy humans.

Human insulin and its synthetic analogs are considered as life-saving drugs for people suffering from diabetes mellitus. Next to the therapeutic use, scientific and non-scientific literature (e.g. bodybuilding forums; antidoping intelligence and investigation reports) indicate that these prohibited substances are used as performance enhancing agents. In the present report, the development and validation of a sensitive analytical strategy is described for the urinary detection of three rapid-acting insulin analogs (Lispro, Aspart, Glulisine). The method is based on sample purification by the combination of ultrafiltration and immunoaffinity purification and subsequent analysis by nano-flow liquid chromatography coupled to high resolution mass spectrometry. Next to the results on different validation parameters (LOD: 10 pg/mL; recovery: 25-48%; matrix effect: -3-(-8) %), data on urinary elimination times, which were obtained in the frame of an administration study with the participation of healthy volunteers, are presented. The determined detection windows (~9 hours) are expected to help to evaluate current routine analytical methods and aim to aid doping authorities to set appropriate target windows for efficient testing.

Preparation and comparison of Fe3O4@graphene oxide nanoclusters for analysis of glimepiride in urine by surface-assisted laser desorption/ionization time-of-flight mass spectrometry.

Graphene oxide (GO) has the ability to absorb certain compounds, and it can be modified with functional groups for different purposes; for instance, iron oxide (IO) nanoparticles can be used to concentrate analyte by a magnet. Recently, many kinds of GO have been developed, such as single-layer GO (SLGO), two-to-four layers of GO (i.e., few-layer GO, FLGO2-4), and four-to-eight layers of GO (i.e., multi-layer GO, MLGO4-8). However, the abilities of these layered GO coated with IO nanoparticles have not been investigated. In this study, we conducted a novel analysis of glimepiride by using layered GO-coated magnetic clusters of IO nanoparticles that were synthesized through a simple and facile emulsion-solvent evaporation method. The methodology is based on (i) enrichment of glimepiride using the layered GO-coated magnetic clusters of IO nanoparticles (IO@SLGO, IO@FLGO2-4, and IO@MLGO4-8), and (ii) rapid determination using magnetic cluster-based surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOFMS). We found that IO@MLGO4-8, the magnetic cluster with the greatest number of GO layers, had the best limit of detection (28.6 pmol/µL for glimepiride). The number of GO layers played a significant role in increasing the sensitivity of the SALDI-MS, indicating that the size of GO in the magnetic clusters contributed to the desorption/ionization efficiency. To the best of our knowledge, this is the first study to enrich glimepiride using magnetic clusters of different GO types and to show that the glimepiride in HLB purified urine adsorbed by magnetic clusters can be analyzed by SALDI-TOFMS.

Simultaneous detection of 24 oral antidiabetic drugs and their metabolites in urine by liquid chromatography-tandem mass spectrometry.

Drugs are the most frequent cause of hypoglycemia. Though the drug history is usually obvious in diabetic patients, the diagnosis could be a challenge in patients without a history of such exposure. Screening for oral antidiabetic drugs has been recommended as part of the hypoglycemia workup in patients without diabetes. Many published analytical methods of oral antidiabetic agents were usually of limited coverage and restricted to parent drugs only. In the current study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical system for the simultaneous detection of 24 oral antidiabetic drugs and their metabolites in urine was established and validated. The method covered both conventional as well as the newer antidiabetic drugs such as dipeptidyl peptidase-4 inhibitors and sodium-glucose cotransporter-2 inhibitors. Following sample preparation by solid phase extraction, analytes were detected by LC-MS/MS with multiple reaction monitoring triggered enhanced product ion scan. The method was successfully applied to 233 cases of unexplained hypoglycemia, with 83 oral antidiabetic drugs detected in 51 of the urine samples.

Square-wave Adsorptive Anodic Stripping Voltammetric Determination of Antidiabetic Drug Linagliptin in Pharmaceutical Formulations and Biological Fluids Using a Pencil Graphite Electrode.

A simple, sensitive, low-cost, quick and reliable square-wave anodic stripping voltammetric method is described for the determination of the antidiabetic drug Linagliptin (LNG) in pure form, tablets, and spiked human urine and plasma samples. Using a pencil graphite electrode (PGE), cyclic voltammetry (CV) was applied to study the electrochemical behavior of LNG. In a Teorell-Stenhagen buffer (pH 5.5) containing 0.1 M NaClO4 as a supporting electrolyte, the LNG yields an irreversible well-defined oxidation peak at about 1.2 V vs. Ag/AgCl electrode. The various affecting factors, such as the pH, buffer type, supporting electrolyte, accumulation potential, scan rate and accumulation time, were tested and optimized. Also, square-wave adsorptive anodic stripping voltammetric (SWAdASV) studies show that the peak current various linearly over the LNG concentration range of 0.24 - 5.20 µg mL-1 (R2 = 0.9994). The detection and quantification limits were calculated to be 0.10 and 0.33 µg mL-1, respectively. The proposed procedure exhibits a good precision, selectivity, and stability and was applied successfully to determine the LNG in pharmaceutical formulations (tablets) and biological fluids (spiked human urine and plasma samples).

Visual and spectrophotometric detection of metformin based on the host-guest molecular recognition of cucurbit[6]uril-modified silver nanoparticles.

A simple, sensitive, and naked-eye assay of metformin (MET), based on the host-guest molecular recognition of cucurbit[6]uril (CB[6])-modified silver nanoparticles, has been developed for the first time. The molecular recognition between CB[6] and MET is initially demonstrated and the related recognition mechanism is further discussed. CB[6]-modified AgNPs were first synthesized and then characterized by UV-vis spectroscopy, Fourier transform infrared spectroscopy, and transmission electron microscopy. The solution behavior of CB[6] in the presence of AgNO3 was also studied, and the correlative result revealed that AgNPs could combine with the carbonyl portals of CB[6]. On the basis of the molecular recognition of CB[6] and the surface plasmon resonance effect of AgNPs, CB[6]-modified AgNPs were used as visual probes to detect MET. In CB[6]-modified AgNP solution, the aggregation of CB[6]-modified AgNPs induced by MET triggered changes of color and the UV-vis absorption spectrum, which laid the foundation for the visual identification and spectrophotometric determination of MET. Under the optimized detection conditions, the UV-vis spectral assay had a good linear relationship in the range from 3 to 750 µM, and the limit of detection was 1 µM. According to the color changes, the minimum concentration recognized by the naked eye was about 75 µM. Furthermore, this assay has high selectivity for coexisting interferents and was also applied to MET detection in human urine samples. This strategy provides a novel and facile tool for highly selective and sensitive detection of MET. Graphical abstract.

Effect of high uric acid on the disposition of metformin: in vivo and in vitro studies.

Metformin is always used as the baseline antidiabetic therapy for patients with type 2 diabetes mellitus (T2DM) and hyperuricemia. Metformin is excreted into urine through active secretion mediated by rOCTs and rMATE1.The aim of this study was to identify the effects of high uric acid on the disposition and its mechanism. For the in vivo study, a hyperuricemic animal model was induced by intraperitoneal injection of potassium oxonate (250 mg/kg) in rats. Metformin (100 mg/kg) was administered orally to investigate the pharmacokinetics in control and hyperuricemic rats, respectively. For the in vitro study, HEK293 and HepaRG cells were used to investigate the effect of uric acid (15 mg/dl) on the expression of OCT1, OCT2 and MATE1 and the disposition of metformin, respectively. The in vivo study showed that the AUC0 â†’ 600 of metformin was significantly decreased by 33.3%, whereas the cumulative urinary excretion of metformin was increased by 25.4% in hyperuricemic rats compared with that in control rats. The renal rOCT1, rOCT2 and rMATE1 and hepatic rMATE1 levels were increased in hyperuricemic rats compared with those in control rats, respectively. The in vitro study showed that uric acid could upregulate the expression of OCT2 and MATE1 in HEK293 cells and MATE1 in HepaRG cells and increase the intracellular metformin concentration in these two cell lines. These results demonstrated that a high uric acid level promoted urinary metformin excretion and decreased the plasma metformin concentration; the in vivo and in vitro studies provided a possible explanation being that high uric acid could upregulate the expression of renal metformin transporters OCTs and MATE1.

A high-performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of imigliptin, its five metabolites and alogliptin in human plasma and urine and its application to a multiple-dose pharmacokinetic study.

Imigliptin is a novel DPP-4 inhibitor, designed to treat type 2 diabetes mellitus (T2DM). A selective and sensitive method was developed using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) to simultaneously quantify imigliptin, its five metabolites, and alogliptin in human plasma and urine. Solid-phase extraction (SPE) and direct dilution were used to extract imigliptin, its five metabolites, alogliptin from plasma and urine, respectively. The extracts were injected onto a SymmetryShield RP8 column with a gradient elution of methanol and water containing 10 mM ammonium formate (pH = 7). Ionization of all analytes was performed using an electrospray ionization (ESI) source in positive mode and detection was carried out with multiple reaction monitoring (MRM) mode. The results revealed that the method had excellent selectivity and linearity. Inter- and intra-batch precisions of all analytes were less than 15% and the accuracies were within 85%-115% for both plasma and urine. The sensitivity, matrix effect, extraction recovery, linearity, and stabilities were validated for all analytes in human plasma and urine. In conclusion, the validation results showed that this method was robust, specific, and sensitive and it can successfully applied to a pharmacokinetic study of Chinese T2DM subjects after oral dose of imigliptin and alogliptin.

Simultaneous determination of 14 oral antihyperglycaemic drugs in human urine by liquid chromatography-tandem mass spectrometry.

A simple, sensitive, and rapid assay based on hydrophilic interaction liquid chromatography (HILIC) with tandem mass spectrometry was developed and validated for the simultaneous determination of metformin and 13 other oral antihyperglycaemic drugs in human urine using metoprolol as an internal standard. A simple sample clean-up procedure using the "dilute and shoot" approach enabled fast and reliable analysis. Chromatographic separation was performed on a HILIC column using an elution gradient of mobile phase A, composed of 1 mM ammonium formate (pH 5), and mobile phase B, composed of acetonitrile, at a flow rate of 0.35 mL/min. Quantitation was performed on a triple quadrupole mass spectrometer operated in multiple reaction monitoring mode by using electrospray ionization in positive ion mode. The total chromatographic run time was 20 min. Calibration curves for each analyte were linear over concentration ranges of 2-300, 5-400, or 20-500 ng/mL, with a coefficient of determination above 0.99. The method was validated for selectivity, sensitivity, recovery, linearity, accuracy and precision, system suitability, robustness, and stability. Inter-batch and intra-batch coefficients of variation across four validation runs were ≤ 13.62%. The present method was successfully applied for the analysis of metformin and nateglinide in urine samples after their oral administration to healthy human subjects under fasted conditions.

A dosing algorithm for metformin based on the relationships between exposure and renal clearance of metformin in patients with varying degrees of kidney function.

PURPOSE: The aims of this study were to investigate the relationship between metformin exposure, renal clearance (CLR), and apparent non-renal clearance of metformin (CLNR/F) in patients with varying degrees of kidney function and to develop dosing recommendations. METHODS: Plasma and urine samples were collected from three studies consisting of patients with varying degrees of kidney function (creatinine clearance, CLCR; range, 14-112 mL/min). A population pharmacokinetic model was built (NONMEM) in which the oral availability (F) was fixed to 0.55 with an estimated inter-individual variability (IIV). Simulations were performed to estimate AUC0-τ, CLR, and CLNR/F. RESULTS: The data (66 patients, 327 observations) were best described by a two-compartment model, and CLCR was a covariate for CLR. Mean CLR was 17 L/h (CV 22%) and mean CLNR/F was 1.6 L/h (69%).The median recovery of metformin in urine was 49% (range 19-75%) over a dosage interval. When CLR increased due to improved renal function, AUC0-τ decreased proportionally, while CLNR/F did not change with kidney function. Target doses (mg/day) of metformin can be reached using CLCR/3 × 100 to obtain median AUC0-12 of 18-26 mg/L/h for metformin IR and AUC0-24 of 38-51 mg/L/h for metformin XR, with Cmax < 5 mg/L. CONCLUSIONS: The proposed dosing algorithm can be used to dose metformin in patients with various degrees of kidney function to maintain consistent drug exposure. However, there is still marked IIV and therapeutic drug monitoring of metformin plasma concentrations is recommended.

Pregnancy Increases the Renal Secretion of N1-methylnicotinamide, an Endogenous Probe for Renal Cation Transporters, in Patients Prescribed Metformin.

N1-methylnicotinamide (1-NMN) has been investigated as an endogenous probe for the renal transporter activity of organic cation transporter 2 (OCT2) and multidrug and toxin extrusion proteins 1 and 2-K (MATE1 and MATE2-K). As pregnancy increased the renal secretion of metformin, a substrate for OCT2, MATE1, and MATE2-K, we hypothesized that the renal secretion of 1-NMN would be similarly affected. Blood and urine samples collected from women prescribed metformin for type 2 diabetes, gestational diabetes, and polycystic ovarian syndrome during early, mid, and late pregnancy (n = 34 visits) and postpartum (n = 14 visits) were analyzed for 1-NMN using liquid chromatography-mass spectrometry. The renal clearance and secretion clearance, using creatinine clearance to correct for glomerular filtration, were estimated for 1-NMN and correlated with metformin renal clearance. 1-NMN renal clearance was higher in both mid (504 ± 293 ml/min, P < 0.01) and late pregnancy (557 ± 305 ml/min, P < 0.01) compared with postpartum (240 ± 106 ml/min). The renal secretion of 1-NMN was 3.5-fold higher in mid pregnancy (269± 267, P < 0.05) and 4.5-fold higher in late pregnancy compared with postpartum (342 ± 283 versus 76 ± 92 ml/min, P < 0.01). Because creatinine is also a substrate of OCT2, MATE1, and MATE2-K, creatinine clearance likely overestimates filtration clearance, whereas the calculated 1-NMN secretion clearance is likely underestimated. Metformin renal clearance and 1-NMN renal clearance were positively correlated (rs = 0.68, P < 0.0001). 1-NMN renal clearance increases during pregnancy due to increased glomerular filtration and net secretion by renal transporters.

Profiling and identification of (-)-epicatechin metabolites in rats using ultra-high performance liquid chromatography coupled with linear trap-Orbitrap mass spectrometer.

(-)-Epicatechin (EC), an optical antipode of (+)-catechin (C), possesses many potential significant health benefits. However, the in vivo metabolic pathway of EC has not been clarified yet. In this study, an efficient strategy based on ultra-high performance liquid chromatography coupled with a linear ion trap-Orbitrap mass spectrometer was developed to profile and characterize EC metabolites in rat urine, faeces, plasma, and various tissues. Meanwhile, post-acquisition data-mining methods including high-resolution extracted ion chromatogram (HREIC), multiple mass defect filters (MMDFs), and diagnostic product ions (DPIs) were utilized to screen and identify EC metabolites from HR-ESI-MS1 to ESI-MSn stage. Finally, a total of 67 metabolites (including parent drug) were tentatively identified based on standard substances, chromatographic retention times, accurate mass measurement, and relevant drug biotransformation knowledge. The results demonstrated that EC underwent multiple in vivo metabolic reactions including methylation, dehydration, hydrogenation, glucosylation, sulfonation, glucuronidation, ring-cleavage, and their composite reactions. Among them, methylation, dehydration, glucosylation, and their composite reactions were observed only occurring on EC when compared with C. Meanwhile, the distribution of these detected metabolites in various tissues including heart, liver, spleen, lung, kidney, and brain were respectively studied. The results demonstrated that liver and kidney were the most important organs for EC and its metabolites elimination. In conclusion, the newly discovered EC metabolites significantly expanded the understanding on its pharmacological effects and built the foundation for further toxicity and safety studies. Copyright © 2017 John Wiley & Sons, Ltd.

Modification on the O-glucoside of Sergliflozin-A: A new strategy for SGLT2 inhibitor design.

Poor pharmacokinetic stability is one of the issues of O-glucoside SGLT2 inhibitors in clinical trials, hence C-glucoside inhibitors have been developed and extensively applied. Herein, we provided an alternative approach to improve the pharmacokinetic stability of such inhibitors. Nine derivatives of Sergliflozin-A with modifications on the O-glucoside fragment were prepared, among which the 4-O-methyl derivative exhibited similar pharmacodynamics potency in excreted glucose urine test. Most attractively, significantly increased pharmacokinetic stability was observed for 4-O-methyl derivative of O-glucosides. This work proved that modification on the O-glucoside fragment could be a promising approach to the future SGLT2 inhibitor design.

Therapeutic effect of atorvastatin on kidney functions and urinary excretion of Glimepiride in healthy adult human male subjects.

Glimepiride and atorvastatin in combination are commonly employed for treating the hyperglycemia and dyslipidemia, respectively, in patients of type 2 diabetes. The present study was designed to find out the influence of atorvastatin on urinary excretion and renal clearance of Glimepiride in healthy adult male volunteers. In each experimental subject, Glimepiride 2mg was given orally after an overnight fasting. Samples of blood and urine were taken at different specific time intervals. After a washout period of ten days, Glimepiride 2mg was co-administered with atorvastatin 20mg orally. Post-medication, blood and urine samples were collected following the same sampling schedule as for Glimepiride alone. The samples were analyzed for Glimepiride and creatinine concentration by HPLC-UV and Spectrophotometer, respectively. Mean (±SE) values for blood pH 7.445±0.05 and 7.382±0.05, urine pH 4.972±0.08 and 5.08±0.10, diuresis 0.0207±0.00 and 0.0237±0.00ml/min/kg, endogenous creatinine in plasma 9.048±0.33 and 8.613±0.024µg/ml, endogenous creatinine in urine 512.34±18.20 and 556.72±4.60µg/ml, Glimepiride plasma concentration 0.16069±0.00 and 0.3227±0.01µg/ml, Glimepiride urine concentration 1.5994±0.03 and 0.8665±0.04µg/ml, renal clearance of creatinine 1.224±0.09 and 1.550±0.09ml/min/kg, renal clearance of Glimepiride 0.2064±0.01 and 0.0641±0.00ml/min/kg and clearance ratio 0.1791±0.01 and 0.0414±0.00 were observed for Glimepiride alone and its concurrent administration with atorvastatin, respectively. Atorvastatin decreased the urinary excretion and renal clearance of Glimepiride due to which chances of hypoglycemia provokes and renal handling of Glimepiride involves back diffusion besides glomerular filtration and no influence of atorvastatin was seen on these mechanisms.

Simultaneous determination of imigliptin and its three metabolites in human plasma and urine by liquid chromatography coupled to tandem mass spectrometry.

A specific and sensitive method was firstly developed using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) to simultaneously quantify imigliptin (KBP-3853) and its three metabolites (KBP-3926, KBP-3902, KBP-5493) in human plasma and urine. Solid-phase extraction (SPE) and direct dilution were used to extract imigliptin and its three metabolites from plasma and urine, respectively. The extracts were injected onto a SymmetryShield RP8 column with a gradient elution of acetonitrile and water containing 5mM ammonium acetate (pH 7). Ionization of KBP-3853, KBP-3926, KBP-3902, KBP-5493, and XZP-3244 (internal standard, IS) was performed using an electrospray ionization (ESI) source in positive mode and detection was carried out with multiple reaction monitoring (MRM) mode. The lower limits of quantitation (LLOQ) of KBP-3853/KBP-3926/KBP-3902/KBP-5493 in human plasma and urine were 0.500/0.500/0.500/0.500ng/mL and 20.0/20.0/10.0/10.0ng/mL, respectively. Inter- and intra-batch precision of imigliptin and its three metabolites were less than 15% and the accuracy was within 85-115% for both plasma and urine. The extraction recoveries of all analytes at three concentration levels were consistent. The specificity, matrix effect, linearity and stabilities under various conditions were validated for imigliptin and its three metabolites in human plasma and urine. In conclusion, the validation results showed that this method was robust, specific, and sensitive and it can successfully fulfill the requirement of clinical pharmacokinetic study of imigliptin hydrochloride in Chinese healthy subjects.

"Dilute-and-inject" multi-target screening assay for highly polar doping agents using hydrophilic interaction liquid chromatography high resolution/high accuracy mass spectrometry for sports drug testing.

In the field of LC-MS, reversed phase liquid chromatography is the predominant method of choice for the separation of prohibited substances from various classes in sports drug testing. However, highly polar and charged compounds still represent a challenging task in liquid chromatography due to their difficult chromatographic behavior using reversed phase materials. A very promising approach for the separation of hydrophilic compounds is hydrophilic interaction liquid chromatography (HILIC). Despite its great potential and versatile advantages for the separation of highly polar compounds, HILIC is up to now not very common in doping analysis, although most manufacturers offer a variety of HILIC columns in their portfolio. In this study, a novel multi-target approach based on HILIC high resolution/high accuracy mass spectrometry is presented to screen for various polar stimulants, stimulant sulfo-conjugates, glycerol, AICAR, ethyl glucuronide, morphine-3-glucuronide, and myo-inositol trispyrophosphate after direct injection of diluted urine specimens. The usage of an effective online sample cleanup and a zwitterionic HILIC analytical column in combination with a new generation Hybrid Quadrupol-Orbitrap® mass spectrometer enabled the detection of highly polar analytes without any time-consuming hydrolysis or further purification steps, far below the required detection limits. The methodology was fully validated for qualitative and quantitative (AICAR, glycerol) purposes considering the parameters specificity; robustness (rRT < 2.0%); linearity (R > 0.99); intra- and inter-day precision at low, medium, and high concentration levels (CV < 20%); limit of detection (stimulants and stimulant sulfo-conjugates < 10 ng/mL; norfenefrine; octopamine < 30 ng/mL; AICAR < 10 ng/mL; glycerol 100 µg/mL; ETG < 100 ng/mL); accuracy (AICAR 103.8-105.5%, glycerol 85.1-98.3% at three concentration levels) and ion suppression/enhancement effects.

Quantitative Evaluation of mMate1 Function Based on Minimally Invasive Measurement of Tissue Concentration Using PET with [(11)C]Metformin in Mouse.

PURPOSE: To evaluate the function of multidrug and toxin extrusion proteins (MATEs) using (11)C-labeled metformin ([(11)C]metformin) by positron emission tomography (PET). METHODS: PET was performed by intravenous bolus injection of [(11)C]metformin. Pyrimethamine at 0.5 and 5 mg/kg was intravenously administered to mice 30 min prior to the scan. Integration plot analysis was conducted for calculating liver (CLuptake,liver), kidney (CLuptake,kidney) tissue uptake, intrinsic biliary (CLint,bile) and urinary (CLint,urine) excretion clearances of [(11)C]metformin. RESULTS: Visualization by PET showed that pyrimethamine increased concentrations of [(11)C]metformin in the liver and kidneys, and decreased the concentrations in the urinary bladder without changing the blood profiles. Pyrimethamine had no effect on the CLuptake,liver and CLuptake,kidney, which were similar to the blood-flow rate. CLint,bile with regard to the liver concentration was unable to be determined, but administration of 0.5 and 5 mg/kg of pyrimethamine increased the liver-to-blood ratio to 1.6 and 2.3-fold, respectively, indicating that pyrimethamine inhibited the efflux of [(11)C]metformin from the liver. CLint,urine with regard to the corticomedullary region concentrations was decreased 37 and 68% of the control by administration of 0.5 and 5 mg/kg of pyrimethamine, respectively (P < 0.05). CONCLUSIONS: Tissue concentration based investigations using [(11)C]metformin by PET enables the functional analysis of MATEs in the liver and kidneys.