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.

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.

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.

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.

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.

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.

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.

Quantitation of metformin in human plasma and urine by hydrophilic interaction liquid chromatography and application to a pharmacokinetic study.

We describe an analytical method for the quantification of the widely used antihyperglycemic agent, metformin, in human plasma and urine. The separation was performed using isocratic hydrophilic interaction liquid chromatography on a Luna hydrophilic interaction liquid chromatography column (125 × 4.6 mm, 3 µm). The sample preparation was accomplished by solid-phase extraction. Validation of the method was performed in the range 10-2000 ng/mL for plasma and 5-30 mcg/mL for urine. The methods were linear within the investigated range (r(2) > 0.988). Within-day repeatability ranged from 3.1% to 7.5% in plasma and 1.6% to 6.2% in urine. Between-day reproducibility ranged from 2.9% to 5.3% in plasma and 0.6% to 1.8% in urine. The inaccuracy expressed as bias ranged from -3.1% to 1.9% in plasma and from -7.2% to 0.7% in urine. The lower limit of quantification for metformin in plasma was 5 ng/mL and in urine was 40 ng/mL. The method was therefore considered to be precise, accurate, reproducible, and sensitive enough to be appropriate for pharmacokinetic studies of metformin. The applicability of the method for human pharmacokinetic studies was demonstrated by dosing a healthy male volunteer with 500-mg metformin hydrochloride as a single oral dose; plasma and urine concentrations were measured for 24 hours.

The effect of lansoprazole, an OCT inhibitor, on metformin pharmacokinetics in healthy subjects.

PURPOSE: Gastro-esophageal reflux disease is common in patients with type 2 diabetes. A common treatment is the co-administration of proton-pump inhibitors (PPIs) and metformin. To date, however, the effects of co-administration of PPIs, which inhibit organic cation transporter (OCT) activity, on the action of metformin (a well-known substrate of OCTs) have not been clearly demonstrated. METHODS: This was a randomized, double-blind, two-way crossover, placebo-controlled trial. Healthy male volunteers (n = 20) received metformin (single dose 1,000 mg on day 1 and single dose 750 mg on day 2, with a 12-h interval) co-administered with placebo or with lansoprazole (30 mg). Plasma concentrations of metformin were measured up to 24 h after the second dose. The glucose-lowering effects of metformin were evaluated by the oral glucose tolerance test before and after each single dose of metformin within the 2-day period. RESULTS: Lansoprazole increased the mean metformin maximum plasma concentration and area under the plasma concentration-time curve from zero to 24 h after the second dosing by 15 and 17 %, respectively (P < 0.05). Moreover, lansoprazole prolonged the metformin elimination half-life from 3.9 to 4.5 h and decreased its renal clearance by 13 % (P < 0.05). However, lansoprazole had no effect on the maximum glucose level and the area under the serum glucose concentration-time curve of metformin. CONCLUSIONS: Collectively, we found a modest pharmacokinetic drug interaction between lansoprazole and metformin, which suggests that the concomitant use of these drugs should be appropriately monitored. Further studies are warranted to assess changes in metformin pharmacokinetics in patients with diabetes receiving long-term lansoprazole therapy.

A gene-gene interaction between polymorphisms in the OCT2 and MATE1 genes influences the renal clearance of metformin.

OBJECTIVE: The aim of this study was to determine the association between the renal clearance (CL(renal)) of metformin in healthy Caucasian volunteers and the single-nucleotide polymorphism (SNP) c.808G>T (rs316019) in OCT2 as well as the relevance of the gene-gene interactions between this SNP and (a) the promoter SNP g.-66T>C (rs2252281) in MATE1 and (b) the OCT1 reduced-function diplotypes. METHODS: Fifty healthy volunteers genotyped for the c.808G>T were enrolled in the study. The distribution was 25 GG, 20 GT, and 5 TT volunteers. The pharmacokinetics of a 500 mg single oral dose of metformin was studied. RESULTS: When analyzed alone, the c.808 (G>T) affected neither the CL(renal) nor the secretory clearance (CL(sec)) of metformin. However, both CL(renal) and CL(sec) were increased for the volunteers with minor alleles in c.808 (G>T) who were also homozygous for the reference variant g.-66T>C: CL(renal): GG, GT, and TT: 28.1, 34.5, and 44.8 l/h (P = 0.004), respectively and CL(sec): GG, GT, and TT: 21.4, 27.8, and 37.6 l/h (P = 0.005), respectively. In the volunteers with minor alleles in c.808 (G>T) who were also heterozygous for g.-66T>C, both CL(renal) and CL(sec) were found to be reduced (P < 0.028) when compared with volunteers with minor alleles in c.808 (G>T) carrying the g.-66T>C reference genotype. CONCLUSION: We report counteracting effects of the c.808 (G>T) and g.-66T>C on the renal elimination of metformin. When adjusted for the genetic variation g.-66T>C, our results suggest that c.808 (G>T) could have a dominant genotype to phenotype correlation.

Competitive inhibition of the luminal efflux by multidrug and toxin extrusions, but not basolateral uptake by organic cation transporter 2, is the likely mechanism underlying the pharmacokinetic drug-drug interactions caused by cimetidine in the kidney.

Cimetidine, an H2 receptor antagonist, has been used to investigate the tubular secretion of organic cations in human kidney. We report a systematic comprehensive analysis of the inhibition potency of cimetidine for the influx and efflux transporters of organic cations [human organic cation transporter 1 (hOCT1) and hOCT2 and human multidrug and toxin extrusion 1 (hMATE1) and hMATE2-K, respectively]. Inhibition constants (K(i)) of cimetidine were determined by using five substrates [tetraethylammonium (TEA), metformin, 1-methyl-4-phenylpyridinium, 4-(4-(dimethylamino)styryl)-N-methylpyridinium, and m-iodobenzylguanidine]. They were 95 to 146 µM for hOCT2, providing at most 10% inhibition based on its clinically reported plasma unbound concentrations (3.6-7.8 µM). In contrast, cimetidine is a potent inhibitor of MATE1 and MATE2-K with K(i) values (µM) of 1.1 to 3.8 and 2.1 to 6.9, respectively. The same tendency was observed for mouse Oct1 (mOct1), mOct2, and mouse Mate1. Cimetidine showed a negligible effect on the uptake of metformin by mouse kidney slices at 20 µM. Cimetidine was administered to mice by a constant infusion to achieve a plasma unbound concentration of 21.6 µM to examine its effect on the renal disposition of Mate1 probes (metformin, TEA, and cephalexin) in vivo. The kidney- and liver-to-plasma ratios of metformin both were increased 2.4-fold by cimetidine, whereas the renal clearance was not changed. Cimetidine also increased the kidney-to-plasma ratio of TEA and cephalexin 8.0- and 3.3-fold compared with a control and decreased the renal clearance from 49 to 23 and 11 to 6.6 ml/min/kg, respectively. These results suggest that the inhibition of MATEs, but not OCT2, is a likely mechanism underlying the drug-drug interactions with cimetidine in renal elimination.

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.

Pharmacokinetics of metformin during pregnancy.

Our objective was to evaluate the pharmacokinetics of metformin during pregnancy. Serial blood and urine samples were collected over one steady-state dosing interval in women treated with metformin during early to late pregnancy (n = 35) and postpartum (n = 16). Maternal and umbilical cord blood samples were obtained at delivery from 12 women. Metformin concentrations were also determined in breast milk samples obtained over one dosing interval in 6 women. Metformin renal clearance increased significantly in mid (723 +/- 243 ml/min, P < 0.01) and late pregnancy (625 +/- 130 ml/min, P < 0.01) compared with postpartum (477 +/- 132 ml/min). These changes reflected significant increases in creatinine clearance (240 +/- 70 ml/min, P < 0.01 and 207 +/- 56 ml/min, P < 0.05 versus 165 +/- 44 ml/min) and in metformin net secretion clearance (480 +/- 190 ml/min, P < 0.01 and 419 +/- 78 ml/min, P < 0.01 versus 313 +/- 98 ml/min) in mid and late pregnancy versus postpartum, respectively. Metformin concentrations at the time of delivery in umbilical cord plasma ranged between nondetectable (<5 ng/ml) and 1263 ng/ml. The daily infant intake of metformin through breast milk was 0.13 to 0.28 mg, and the relative infant dose was <0.5% of the mother's weight-adjusted dose. Our results indicate that metformin pharmacokinetics are affected by pregnancy-related changes in renal filtration and net tubular transport and can be roughly estimated by the use of creatinine clearance. At the time of delivery, the fetus is exposed to metformin concentrations from negligible to as high as maternal concentrations. In contrast, infant exposure to metformin through the breast milk is low.

Determination of metformin in human plasma and urine by high-performance liquid chromatography using small sample volume and conventional octadecyl silane column.

PURPOSE: To develop a selective and sensitive high-performance liquid chromatographic method for the determination of metformin in human plasma and urine, using a conventional reverse phase column and low specimen volume. METHODS: Extraction of metformin and ranitidine (as internal standard) from plasma and urine samples (100 µL) was performed with a 1-butanol-hexane (50:50, v/v) mixture under alkaline conditions followed by back-extraction into diluted acetic acid. Chromatography was carried out using a C18 column (250 mm×4.6 mm, 5 µm). A mobile phase consisting of acetonitrile and KH2PO4 (34:66, v/v) and sodium dodecyl sulphate (3 mM) was pumped at an isocratic flow rate of 0.7 mL/min. RESULTS: The calibration curves were linear (>0.995) in the concentration ranges of 10-5000 and 2-2000 µg/mL for metformin in plasma and urine respectively. .The mean absolute recoveries for 100 and 1000 ng/mL metformin in plasma using the present extraction procedure were 93.7 and 88.5%, respectively. The intra- and inter-day coefficients of variation in plasma and urine were <20% at the lowest, and <16% at other concentrations. The percent error values were less than 2% in plasma while it reached ~9% in urine. The lower limits of quantification were 7.8 ng/mL and 1.6 µg/mL of metformin base in plasma and urine respectively. CONCLUSION: The method showed high calibers of sensitivity and selectivity for monitoring therapeutic concentrations of metformin in both plasma and urine based on a 0.1 ml sample size._____________________________________________________________________________________

Optimization and validation of an RP-HPLC method for direct determination of metformin hydrochloride in human urine and in a dosage form.

A simple, selective, sensitive, accurate, and precise method was developed for determination of metformin hydrochloride (MF) in human urine using RP-HPLC. The method depends upon using an octylsilyl (C8) 5 microm particle size column at ambient temperature with mobile phase consisting of 33 mM sodium dihydrogen phosphate containing 6.38 mM hexanesulfonic acid sodium salt and adjusted to apparent pH 3.0 with phosphoric acid-acetonitrile (93 + 7, v/v) at a flow rate of 1.5 mL/min. Quantitation was achieved with UV detection at 231 nm based on peak area with a linear calibration curve over the concentration range of 0.01-50 microg/mL. The proposed method was applied to the determination of the urinary excretion pattern of MF (the cumulative amounts excreted were calculated without pretreatment of the urine sample) and for determination of the dissolution pattern of MF tablets. The proposed method was completely validated according to the U.S. Food and Drug Administration guidelines.