Rapid detection of the metformin illegally added of in TCM and health food by TLC-IR.

Based on TLC-IR, the study established an effective method for rapid detection of metformin illegally added in hypoglycemic traditional Chinese medicine and health food products. 12 batches of hypoglycemic traditional Chinese medicine and health products were purchased in the pharmacy, which were produced by different manufacturers. TLC was used to separate metformin and phenformin for preliminary identification from. IR was applied to further identification and HPLC method was used to verify the experimental results of TLC-IR. TLC developing solvents was petroleum ether-methanol-glacial acetic acid (5:12:0.5) and the stationary phase was silica gel prefabricated GF254 plate. IR used KBr pellet pressing method with a resolution of 4cm-1 and scanned 64 times. The column for HPLC analysis was SinoChrom ODS-BP 5 µm (4.6mm *250mm) and the injection volume was 20µL. The detection wavelength was 234nm. The flow rate was 1ml•min-1. Metformin and phenformin were significantly separated under the TLC condition. Joint identification by TLC-IR, none of metformin and phenformin were identified in the hypoglycemic traditional Chinese medicine. Phenformin was detected in two kinds of health products while metformin was identified in one kind of health food. The result of HPLC was consist with TLC-IR. The established TLC-IR method was simple, rapid and selective, which was suit to apply in the identification of metformin illegally added in hypoglycemic traditional Chinese medicine and health food products.

Highly sensitive detection of an antidiabetic drug as illegal additives in health products using solvent microextraction combined with surface-enhanced Raman spectroscopy.

A rapid and accurate method for the sensitive detection of illegal drug additives including atenolol (ATN), metformin hydrochloride (MET), and phenformin hydrochloride (PHE) in health products using solvent microextraction (SME) combined with surface-enhanced Raman spectroscopy (SERS) was developed. Various illegal drug additives in different health products were separated via microextraction and then detected in situ using a portable Raman spectrometer with Ag colloids acting as SERS-active substrates. The effects of experimental parameters on the detection sensitivity and producibility were evaluated, and the applications of illegal additives spiked into samples were systematically investigated with SME-SERS. It was demonstrated that the mixture of CH3OH and CHCl3 (v/v = 1 : 4) as the extractant was suitable for the rapid microextraction separation of illegal drug additives and also induced the distribution of the Ag colloids (2 M) on the CHCl3 surface. More importantly, CH3OH can carry the drug molecules to enter into the inter-particles of the Ag colloids in this process, and then significantly improve the detection sensitivity of illegal drug additives. Furthermore, the high-throughput and real-time detection of illegal drug additives spiked into health products with SME-SERS in multi-well 96 plates were achieved with the level of 0.1 µg mg-1. The results reveal that this rapid and convenient method could be used for the effective separation and sensitive detection of illegal additives in complex specimen.

Combined host-guest complex with coffee-ring effect for constructing ultrasensitive SERS substrate for phenformin hydrochloride detection in healthcare products.

Phenformin hydrochloride (PHE), once used as a traditional anti-diabetic drug, has now been banned due to significant side effects. However, the phenomenon of the illegal addition of PHE to hypoglycemic healthcare products is still rampant. Thus, the detection of illegally added PHE is urgently needed. Surface-enhanced Raman scattering (SERS) is a promising candidate for this purpose, but the weak affinity between PHE and bare metal (Au or Ag) limits direct SERS detection of PHE. In this paper, we prepared Ag nanoparticles coated with ß-cyclodextrin (AgNP@ß-CD), which display the coffee-ring effect, that can be used for PHE sensing. ß-CD-functionalized nanoparticles could capture the analyte and fix the molecular orientation in the hydrophobic cavity. The coffee-ring effect could improve the SERS effect through a higher concentration of the analyte, higher density of nanoparticles, and more hot spots. The SERS performance of the AgNP@ß-CD substrate was characterized by using o-phenylenediamine dihydrochloride as a probe molecule. The excitation wavelength and pH value were optimized. A linear response for PHE detection is in the 7.0 × 10-8-1.0 × 10-6 mol L-1 concentration range, and the limit of detection is as low as 8.0 × 10-9 mol L-1. This AgNP@ß-CD coffee-ring effect substrate was applied to the detection of PHE in healthcare products, with recoveries between 95.3 and 105.0% and relative standard deviations of less than 5.16%. It is anticipated that the AgNP@ß-CD substrate will also have great potential for the monitoring of other aromatic drugs in healthcare products.

Determination of phenformin hydrochloride employing a sensitive fluorescent probe.

A complexation of non-fluorescent phenformin hydrochloride (PFH) with cucurbit [7]uril (CB [7]) in aqueous solution was investigated using the fluorescent probe of palmatine (PAL) coupled with CB [7]. The fluorescent probe of CB [7]-PAL exhibited strong fluorescence in aqueous solution, which was quenched gradually with the increase of PFH. This effect is observed because when PFH was added to the host-guest system of CB [7]-PAL, PFH and PAL competed to occupy the CB [7] cavity. Portions of the PAL molecule were expelled from the CB [7] cavity owing to the introduction of PFH. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescence method of high sensitivity and selectivity was developed to determine PFH with good precision and accuracy for the first time. The linear range of the method was 0.005-1.9 µg mL(-1) with a detection limit of 0.003 µg mL(-1). In this work, association constants (K) of PFH with CB [7] were also determined. KCB [7]-PFH=(2.52±0.05)×10(5) L mol(-1). The ability of PFH to bind with CB [7] is stronger than that of PAL. The results of a density functional theory calculation authenticated that the moiety of PFH was embedded in the hydrophobic cavity of CB [7] tightly, and the nitrogen atom is located in the vicinity of a carbonyl-laced portal in the energy-minimized structure. The molecular modelling of the interaction between PFH and CB [7] was also confirmed by (1)H NMR spectra (Bruker 600 MHz).

Graphene and CdS nanocomposite: a facile interface for construction of DNA-based electrochemical biosensor and its application to the determination of phenformin.

Graphene/cadmium sulphide (GR-CdS) nanocomposite was synthesized via a low temperature process in aqueous solution. The as-prepared nanocomposite was characterized by scanning electron microscopy, UV-visible spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction. The impedance analysis indicated that GR-CdS nanocomposite possessed outstanding electrochemical performance for facile electron transfer. When DNA was immobilized on GR-CdS (DNA/GR-CdS) modified electrode, the electrochemical oxidation of guanine and adenine in DNA residue bases was significantly promoted. Due to the interaction of DNA with phenformin, the voltammetric current of guanine or adenine on the DNA/GR-CdS electrode was decreased when phenformin was present in the electrolytic solution. Under optimized conditions, the signal of guanine on DNA/GR-CdS electrode decreased linearly with increasing the concentration of phenformin in the range of 1.0×10(-6)molL(-1) to 1.0×10(-3)molL(-1). The proposed DNA-based electrochemical biosensor was successfully applied to the determination of phenformin in real samples.

Simultaneous determination and pharmacokinetic study of metformin and rosiglitazone in human plasma by HPLC-ESI-MS.

A fast, sensitive and specific high-performance liquid chromatography tandem mass spectrometry method was developed for simultaneous determination of metformin and rosiglitazone in human plasma. With phenformin as an internal standard, the analysis was carried out on a C(18) column (50 mm × 2.1 mm, 3.5 µm) using a mobile phase consisting of acetonitrile-10 mM ammonium acetate (20:80, v/v). The detection was performed by tandem mass spectrometry via electrospray ionization. Linear calibration curves were obtained in the concentration of 1.054-263.5 ng/mL for rosiglitazone and 4.040-5050 ng/mL for metformin. The method was applicable to clinical pharmacokinetic study of metformin and rosiglitazone in healthy volunteers following oral administration.

Electrodeless, accurate pH determination in highly basic media using a new set of (1)H NMR pH indicators.

A set of indicator molecules was selected and applied to elaborate an NMR-based pH determination method, free of glass electrode errors in highly basic media. Accurate measurement of pH values and protonation constants was achieved by a successive build-up of overlapping, increasingly high pH solutions, using a collection of 8 compounds of appropriately incremented basicities. In order to verify the method, acid-base properties were quantified for two compounds with very high basicities in conflicting reports: two pharmaceutically important biguanidine drugs, metformin and phenformin.

N-bromosuccinimide-fluorescein based sensitive flow-injection chemiluminescence determination of phenformin.

A novel and highly sensitive method for the determination of phenformin over the range of 6 x 10(-9) - 1 x 10(-5) g ml(-1) in pharmaceutical formulations with flow-injection chemiluminescence (CL) detection is proposed. The method is based on the CL produced during the oxidation of N-bromosuccinimide (NBS) in an alkaline medium in the presence of fluorescein as an effective energy transfer agent. The use of cetyltrimethylammonium bromide (CTAB) as a sensitizer enhances the signal magnitude by about 100 times. The detection limit is 2 x 10(-9) g ml(-1) (3sigma) with a relative standard deviation of 2.3% (n = 11) at 1 x 10(-7) g ml(-1) phenformin. Ninety samples can be determined per hour. The method was evaluated by carrying out a recovery study and by the analysis of commercial formulations. The obtained results compared well with those by an official method, and demonstrated good accuracy and precision. The possible CL mechanism of the proposed system was also briefly analyzed.

Study of phenformin metabolism in rat liver microsomes by HPLC, CE and on-line HPLC-electrospray ionization mass spectrometry.

Methods for the analysis of phenformin and its metabolite by high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) are developed. The effects of pH, buffer concentration and proportion of organic modifier on the retention of the compounds in HPLC have been studied. The optimum condition was used for the separation and identification of phenformin and its metabolite in microsomal metabolism by HPLC-ESIMS. A simple CE method is also described for the separation of these compounds. Optimum incubation conditions and cofactor requirements for the formation of 4-hydroxyphenformin by microsomal preparations of rat liver were determined. A linear response in the formation of product was found with increasing concentrations of protein and up to 15 min incubation. High concentrations of phenformin inhibited its metabolite formation, and K(m) was 4 microM.

Determination of oral anti-diabetic agents in human body fluids using high-performance liquid chromatography.

Two widely prescribed anti-diabetic agents for which no simple assay method was previously available can now be determined by high-performance liquid chromatography using a UV detection system. The two drugs investigated were tolbutamide (a sulphonylurea) and phenformin (a biguanide). Tolbutamide can be assayed directly, after a single extraction step, on a reversed-phase system, illustrating the simplicity of the technique for carrying out analyses on underivatised drug compared with gas chromatography. Phenformin was not so easily chromatographhed using straightforward partition systems; however, by the choice of a suitable ion-pair agent it was possible to chromatograph the underivatised drug in a relatively simple reversed-phase system.

Characterization of phenformin and metabolites in plasma.

A colorimetric assay of biguanides was adapted for small volumes of plasma and its specificity was improved. This method is based on the reaction of guanidine groups with alpha-naphtol-diacetyl. Interference of endogenous guanidine derivatives and of the water-soluble metabolites of phenformin can be excluded by the extraction procedure. Counting of plasma fractions from 14C-phenformin-injected rats and thin-layer chromatography, before and after treatment with beta-glucuronidase, were also performed: the results suggest that after adequate extraction of plasma, the colorimetric assay measures specifically the biologically active phenformin. Results of this assay in plasma from biguanide-induced lactic acidotic patients and rats are given and compared with controls : results are consistent with the hypothesis of an accumulation of biologically active biguanide in such cases.

Gas chromatographic determination of beta-phenethylbiguanide and its metabolite p-hydroxy-beta-phenethylbiguanide in serum and urine.

A gas chromatographic method is presented for the detection of beta-phenethylbiguanide (PEBG) and its metabolite, p-hydroxy-beta-phenethylbiguanide (p-OHPEBG). The procedure is applicable for the determination of the drug and its metabolite in the serum and urine of rats. The detection limit is 0.2 mug PEBG and 0.5 mug p-OH PEBG per ml of serum or urine. A time course study of blood concentration and elimination rate following intraperitoneal injection of 100 mg/kg of PEBG to normal rats was performed. Beta-PEBG was found to be present in the blood and the urine, p-OH PEBG was only detected in the urine. Twenty-four hours following intraperitoneal injection, the urine contained 32% of the administered dose, 20% as unaltered PEBG and 12% as p-OH PEBG.