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.

Surface molecularly imprinted polymer on magnetic multi-walled carbon nanotubes for selective recognition and preconcentration of metformin in biological fluids prior to its sensitive chemiluminescence determination: Central composite design optimization.

Novel molecularly imprinted polymer (MIP) for metformin was synthesized on the surface of magnetic multi-walled carbon nanotubes (MMWCNTs) as the support. Metformin was used as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker and 2,2'-azoisobutyronitrile (AIBN) as the initiator. The synthesized composite was characterized by field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), and Fourier transform infrared spectroscopy (FTIR). The surface molecularly imprinted composite was used for magnetic solid phase microextraction (MSPME) of metformin before its chemiluminescence (CL) determination and its capability was compared with non-imprinted polymer (NIP). The central composite design was used for optimization as well as consideration of possible interaction of effective variables on extraction. Under the optimized conditions, the developed method exhibited the linear dynamic range of 0.5-50.0 µg L-1 with a detection limit of 0.13 µg L-1 and enhancement factor of 195.3 for the preconcentration of 100 mL of the sample and 500 µL of an eluent. The intra- and inter-day relative standard deviations (RSD%) at 5.0 µg L-1 level of metformin (n = 6) were 3.7 and 4.9%, respectively. The maximum adsorption capacity of the sorbent was found to be 80.0 mg g-1, the adsorption of metformin was endothermic and spontaneous and followed the Langmuir isotherm model. The adsorption kinetic was also found to be best fitted with the pseudo-second-order model. The designed method was successfully applied to the extraction and determination of metformin in biological fluids and water 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.

Alteration of renal excretion pathways in gentamicin-induced renal injury in rats.

The kidney plays a major part in the elimination of many drugs and their metabolites, and drug-induced kidney injury commonly alters either glomerular filtration or tubular transport, or both. However, the renal excretion pathway of drugs has not been fully elucidated at different stages of renal injury. This study aimed to evaluate the alteration of renal excretion pathways in gentamicin (GEN)-induced renal injury in rats. Results showed that serum cystatin C, creatinine and urea nitrogen levels were greatly increased by the exposure of GEN (100 mg kg-1 ), and creatinine concentration was increased by 39.7% by GEN (50 mg kg-1 ). GEN dose-dependently upregulated the protein expression of rOCT1, downregulated rOCT2 and rOAT1, but not affected rOAT2. Efflux transporters, rMRP2, rMRP4 and rBCRP expressions were significantly increased by GEN(100), and the rMATE1 level was markedly increased by GEN(50) but decreased by GEN(100). GEN(50) did not alter the urinary excretion of inulin, but increased metformin and furosemide excretion. However, GEN(100) resulted in a significant decrease of the urinary excretion of inulin, metformin and p-aminohippurate. In addition, urinary metformin excretions in vivo were significantly decreased by GEN(100), but slightly increased by GEN(50). These results suggested that GEN(50) resulted in the induction of rOCTs-rMATE1 and rOAT3-rMRPs pathway, but not changed the glomerular filtration rate, and GEN(100)-induced acute kidney injury caused the downregulated function of glomerular filtration -rOCTs-rMATE1 and -rOAT1-rMRPs pathway.

The urinary excretion of metformin, ceftizoxime and ofloxacin in high serum creatinine rats: Can creatinine predict renal tubular elimination?

The renal excretion of creatinine and most drugs are the net result of glomerular filtration and tubular secretion, and their tubular secretions are mediated by individual transporters. Thus, we hypothesized that the increase of serum creatinine (SCr) levels attributing to inhibiting tubular transporters but not glomerular filtration rate (GFR) could be used to evaluate the tubular excretion of drugs mediated by identical or partial overlap transporter with creatinine. In this work, we firstly developed the creatinine excretion inhibition model with normal GFR by competitively inhibiting tubular transporters, and investigated the renal excretion of metformin, ceftizoxime and ofloxacin in vivo and in vitro. The results showed that the 24-hour urinary excretion of metformin and ceftizoxime in model rats were decreased by 25% and 17% compared to that in control rats, respectively. The uptake amount and urinary excretion of metformin and ceftizoxime could be inhibited by creatinine in renal cortical slices and isolated kidney perfusion. However, the urinary excretion of ofloxacin was not affected by high SCr. These results showed that the inhibition of tubular creatinine transporters by high SCr resulted to the decrease of urinary excretion of metformin and ceftizoxime, but not ofloxacin, which implied that the increase of SCr could also be used to evaluate the tubular excretion of drugs mediated by identical or partial overlap transporter with creatinine in normal GFR rats.

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.

Different effect of testosterone and oestrogen on urinary excretion of metformin via regulating OCTs and MATEs expression in the kidney of mice.

The aim of this study was to investigate the effect of testosterone and oestrogen on regulating organic cation transporters (Octs) and multidrug and toxin extrusions (Mates) expression in the kidney of mice and urinary excretion of metformin. 8 week-old male db/db mice were treated with estradiol (5 mg/kg), testosterone (50 mg/kg) or olive oil with same volume. Metformin (150 mg/kg) was injected in daily for successive 7 days. Plasma, urine and tissue concentrations of metformin were determined by liquid chromatography-tandem mass spectrometry (LCMS) assay. Western blotting and Real-time PCR analysis were successively used to evaluate the renal protein and mRNA expression of Octs and MATEs. After treatment, the protein expression of Mate1 and Oct2 in testosterone group was significantly increased than those in control group (both P < 0.05). The protein expression of Mate1 and Oct2 in estradiol group was significantly reduced by 29.4% and 43.3%, respectively, compared to those in control group (all P < 0.05). These data showed a good agreement with the change in mRNA level (all P < 0.05). The plasma metformin concentration (ng/ml) in mice treated with estradiol was significantly higher than control and testosterone group (677.56 ± 72.49 versus 293.92 ± 83.27 and 261.46 ± 79.45; P < 0.01). Moreover, testosterone increased the metformin urine excretion of mice while estradiol decreasing (both P < 0.01). Spearman correlation analysis showed that gonadal hormone was closely associated with Mate1 and Oct2 expression and metformin urine excretion in db/db mice (all P < 0.05). Testosterone and oestrogen exerted reverse effect on metformin urinary excretion via regulating Octs and Mates expression in the kidney of mice.

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.

N(1)-methylnicotinamide as an endogenous probe for drug interactions by renal cation transporters: studies on the metformin-trimethoprim interaction.

PURPOSE: N(1)-methylnicotinamide (NMN) was proposed as an in vivo probe for drug interactions involving renal cation transporters, which, for example, transport the oral antidiabetic drug metformin, based on a study with the inhibitor pyrimethamine. The role of NMN for predicting other interactions with involvement of renal cation transporters (organic cation transporter 2, OCT2; multidrug and toxin extrusion proteins 1 and 2-K, MATE1 and MATE2-K) is unclear. METHODS: We determined inhibition of metformin or NMN transport by trimethoprim using cell lines expressing OCT2, MATE1, or MATE2-K. Moreover, a randomized, open-label, two-phase crossover study was performed in 12 healthy volunteers. In each phase, 850 mg metformin hydrochloride was administered p.o. in the evening of day 4 and in the morning of day 5. In phase B, 200 mg trimethoprim was administered additionally p.o. twice daily for 5 days. Metformin pharmacokinetics and effects (measured by OGTT) and NMN pharmacokinetics were determined. RESULTS: Trimethoprim inhibited metformin transport with K i values of 27.2, 6.3, and 28.9 µM and NMN transport with IC50 values of 133.9, 29.1, and 0.61 µM for OCT2, MATE1, and MATE2-K, respectively. In the clinical study, trimethoprim increased metformin area under the plasma concentration-time curve (AUC) by 29.5 % and decreased metformin and NMN renal clearances by 26.4 and 19.9 %, respectively (p ≤ 0.01). Moreover, decreases of NMN and metformin renal clearances due to trimethoprim correlated significantly (r S=0.727, p=0.010). CONCLUSIONS: These data on the metformin-trimethoprim interaction support the potential utility of N(1)-methylnicotinamide as an endogenous probe for renal drug-drug interactions with involvement of renal cation transporters.

Sequential hollow-fiber liquid phase microextraction for the determination of rosiglitazone and metformin hydrochloride (anti-diabetic drugs) in biological fluids.

A new analytical method for the simultaneous determination of the antidiabetic drugs rosiglitazone (ROS) and metformin hydrochloride (MH) with marked differences in their affinity towards organic solvents (log P of 2.4 and -1.43, respectively) was developed. Prior to the HPLC separation, the drugs were subjected to a sequential hollow fiber liquid phase microextraction (HF-LPME) procedure. Two sequential HF-LPME approaches were considered, the preferred one involves the use of two vials containing solution mixtures for the extraction of ROS (vial 1) and MH (vial 2), respectively, but using the same fiber and acceptor phase. Important parameters that affect the extraction efficiency such as extracting solvent, donor phase conditions, HCl concentration, agitation, extraction time, addition of salt, etc. were studied. Under the optimum conditions, good enrichment factors (EF, 471 and 86.6 for ROS and MH, respectively) were achieved. Calibration curves were linear over the range 1-500 (r(2)=0.998) and 5-2500 ng mL(-1) (r(2)=0.999) for ROS and MH, respectively. The relative standard deviation values (RSD%) for six replicates were below 8.4%. Detection and quantitation limits based on S/N ratio of 3 and 10 were 0.12, 1.0 and 0.36, 3.0 ng mL(-1) for ROS and MH, respectively. The proposed method is simple, sensitive and opens up new opportunities for the microextraction of analytes with contrasting properties.

Inhibitory effect of atenolol on urinary excretion of metformin via down-regulating multidrug and toxin extrusion protein 1 (rMate1) expression in the kidney of rats.

Renal tubular secretion is an important pathway for the elimination of many clinically used drugs. Metformin, a commonly prescribed first-line antidiabetic drug, is secreted primarily by the renal tubule. Many patients with type 2 diabetes mellitus (T2DM) receiving metformin may together be given selective ß1 blockers (e.g., atenolol). Therefore, it is of great use to evaluate the effect of atenolol on metformin urinary excretion for exploring drug interactions and predicting the adverse effect of drugs. The aim of this study was to investigate the effect of atenolol on the pharmacokinetic of metformin and plasma lactate (LCA) level in rats, for high LCA is a serious adverse reaction of metformin after long-term metformin treatment. In this study, rats were treated with metformin alone or in combination with atenolol. Plasma, urine and tissue concentration of metformin was determined by HPLC method, while Western blotting and immunohistochemical analysis were used to evaluate the renal expression of rat organic cation transporter 2 (rOct2) and multidrug and toxin extrusion protein 1 (rMate1). The results showed that, after 7 days drug treatment, the AUC0 → t of metformin in atenolol and metformin co-administration group was significantly increased by 19.5% compared to that in metformin group, while the 24h cumulative urinary excretion of metformin was significantly decreased by 57.3%. In addition, atenolol treatment significantly decreased the renal expression of rMate1, but had no effect on rOct2 expression, renal blood perfusion and glomerular filtration. Moreover, plasma LCA level in atenolol and metformin co-administration group was significantly increased by 83.3% compared to that in metformin group after 60 days drug treatment. These results indicated that atenolol can inhibit urinary excretion of metformin via decreasing renal rMate1 expression, and long-term atenolol and metformin co-administration may induce potential lactic acidosis. Our results, for the first time, provided an important experimental evidence that rMate1 is the target of transporter-mediated drug interactions concerning metformin and atenolol.

Role of age-related decrease of renal organic cation transporter 2 in the effect of atenolol on renal excretion of metformin in rats.

Many diabetes patients, especially the elder ones, suffered from hypertension simultaneously. Therefore, it is very likely that a large number of diabetes patients receiving metformin hydrochloride may simultaneously be given beta-blockers. Knowing that both metformin and atenolol are eliminated by organic cation transporter 2 (OCT2/SLC22A2) expressed in the renal basolateral membrane, it is not clear whether there is a competitive effect on the renal excretion of metformin and/or atenolol when metformin and atenolol were co-administered, and whether age was involved in this drug-drug interaction. In this present study, both young rats (aged 3 months) and aged rats (aged 12 months) were used, rats were divided into metformin-treated group and metformin and atenolol co-administrated group, respectively. Either metformin (2.5 mg/kg) alone or metformin (2.5 mg/kg) in combination with atenolol (8 mg/kg) was administered to rats by tail vein injection. Then, urine was collected and the metformin concentration in urine was determined by HPLC. The localization and expression of rOCT2 in kidney were also investigated by Western blotting and immunohistochemistry. Significant differences of t 1/2, K e, CLtot and the accumulated metformin excretion in urine were founded in aged rats, but not in young rats, between metformin-treated group (2.002 ± 0.51 h, 0.346 ± 0.07/h, 57.161 ± 18.59 %, 4,287.087 ± 458.08 µg) and metformin plus atenolol-treated group (3.03 ± 0.67 h, 0.228 ± 0.05/h, 43.199 ± 10.28 %, 3,239.972 ± 446.61 µg). Moreover, a significant age-related decrease in rOCT2 protein expression was observed in the aged rats (P < 0.01), which may be responsible for the effect of atenolol on the renal excretion of metformin in the aged rats. In conclusion, there is a drug-drug interaction between atenolol and metformin, and more attention should be paid when atenolol and metformin were co-administered to the aged people inclinical.

Quantitative determination of metformin, glyburide and its metabolites in plasma and urine of pregnant patients by LC-MS/MS.

This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87-99%, and that from urine samples was 85-95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100-0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984-1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy was 86-114% in plasma, and 94-105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine.

Synthesis of Fe-Cu/TiO2 nanostructure and its use in construction of a sensitive and selective sensor for metformin determination.

A carbon paste electrode modified with Fe-Cu/TiO2 was prepared and used for low level determination of metformin (MET) using square wave adsorptive stripping voltammetry (SWAdSV). The Fe-Cu/TiO2 nanoparticle was synthesized by a modified sol-gel method. The surface structure and composition of nanoparticles were characterized by scanning electron microscopy (SEM), X-ray powder diffraction analysis (XRD) and N2 physisorption. Also, electrochemical properties of the prepared nanocomposite modified electrode were investigated by cyclic voltammetry and electrochemical impedance spectroscopy (EIS) techniques. Under optimized conditions, the modified electrode exhibited a linear response over the concentration range of 15 nM to 75 µM MET, with a detection limit of 3 nM. The proposed sensor exhibited a high sensitivity, good selectivity and was successfully applied for MET determination in real samples such as human urine and pharmaceutical formulations.

Large volume sample stacking for rapid and sensitive determination of antidiabetic drug metformin in human urine and serum by capillary electrophoresis with contactless conductivity detection.

Two CE methods with contactless conductivity detection have been developed for determining the oral antidiabetic drug metformin in human urine and blood. The determination of metformin is performed on a separation capillary with an effective length of 14 cm, using a maximum voltage of 30 kV and with a small injection of 50-fold diluted urine into the capillary. Under these conditions, the migration time of metformin is 35s and the LOD is 0.3 µM. Large-volume sample stacking was used to determine low metformin levels in serum. The injection of a sample of serum deproteinized with acetonitrile was 10 times greater compared to the injected amount of urine. This enabled reduction of the LOD to 0.03 µM and the metformin migration time equalled 86 s. The undesirable solvent from sample zone was forced out of the capillary to ensure rapidity and good repeatability of the determination. The RSD values for the migration time are 0.1% for urine and 0.7% for serum; RSD for the peak areas equalled 1.4% for urine and 2.6% for serum. The developed CE technique was tested on performance of routine analyses of metformin in the urine and serum of patients suffering from type II diabetes mellitus.