Microgravimetric method for the determination of metformin in pharmaceutical preparations and in the bulk drug

This article reports a microgravimetric method for the quantitative determination of metformin in bulk and in pharmaceutical preparations. The analysis is simple and relatively rapid. A complete analysis can be performed in only an hour and thirty minutes due to the drying procedure of the precipitate. However a sample can be treated every each five minutes. The method is based on the reaction of nickel (II) with metformin that in basic medium forms an orange precipitate. The reaction is performed in a plastic syringe. The filtration was done in a glass tube containing a sintered glass disk or in Millipore® syringe filters, with posterior weighing after drying. The analytical results were statistically compared with that obtained with a HPLC and with the titrimetric method suggested by the United States and by the Japanese pharmacopoeias.


INTRODUCTION
Diabetes mellitus (DM) is a heterogeneous syndrome characterized by endocrine-metabolic abnormalities that modify the metabolic homeostasis in humans [1].This pathology has been in continuous increasing and among the chronicle non transmissible diseases it is one of the more relevant [2].
Metformin chloridrate is a white crystalline solid.Its molecular formula is C 4 H 11 N 5 •HCl (Fig. 1) and the molecular mass 165.63 Da.Is freely soluble in water but insoluble in acetone, ether and chloroform [4,5].

Figure 1
Some analytical procedures are reported in the literature for the determination of metformin [6,7].
An official analytical method of metformin chloridrate indicated in various pharmacopoeias is the potentiometric titration in non aqueous solution [8][9][10][11].Conductimetric titrations are proposed by Abou-Dan et al. [12] Catalayud et al. [13].and Sartorι et al. [14].Spectrophotometric methods for the determination in bulk drug and in pharmaceutical preparations have been also reported.In the ultraviolet region methods some are proposed: British Pharmacopoeia [10], Annapurna et al. [15], Ashour and Kabbani [16].In the visible region a method was related by Hassan et al. [17].
This last method was based of the colored complex formed between metformin (MF) and copper (II) with posterior dissolution in an organic solvent.
Chromatographic HPLC procedures present good selectivity due to the separation in columns but present an environmental disadvantage that is the relatively high volume of organic solvents used.Despite this fact, it can be satisfactorily used for the simultaneous analysis of metformin and other drugs present in the same preparations [18,[23][24][25][26][27][28][29][30][31][32][33].
Recently Tubino et al. related an analytical quantitative method for the determination of metformin in pharmaceutical preparations and in the bulk drug using diffuse ultraviolet-visible reflectance [34].
Despite the reporting of a number of methods for the analysis of metformin, a deficiency is observed with respect to absolute methods, i.e., that do not require calibration curves.Only three volumetric [12][13][14] are reported and no one for gravimetric was found.
Despite their absolute analytical characteristic classical gravimetric methods are usually very slow.However, the developing of the weighing techniques that resulted in better and faster to use balances, relatively fast gravimetric procedures can be developed [35].
The aim of the present work was to develop a reliable, simple and relatively fast method for the determination of metformin in bulk drugs and in pharmaceutical preparations.

Reagents
All the employed reagents were of analytical grade, excepting the metformin hydrochloride that was a pharmaceutical grade 98.5%, certified product.The pharmaceutical preparations were purchased in drugstores of the region.

Solutions
Standard solution of metformin hydrochloride: prepared in calibrated 50 mL volumetric flask; about 7.5 grams (weighed to 0.1 mg) of the drug (98.5 % certified metformin hydrochloride) was dissolved in Milli Q Pure class water.
Nickel chloride 1 mol L -1 solution: 11.9 g of NiCl 2 •6H 2 O were dissolved with Milli Q Pure grade water in a 50 mL volumetric flask.
NaOH 2 mol L -1 solution: 4.0 g of NaOH were dissolved in Milli Q Pure grade water in a 50 mL volumetric flask.
HClO 4 0.1 mol L -1 in glacial acetic acid [11]: 8.5 mL of perchloric acid were dissolved in 500 mL of glacial acetic acid; 21 mL of acetic anhydride were added; the volume was completed to 1000 mL with glacial acetic acid.The solution was standardized with potassium biphthalate.
Standard (NH 4 )H 2 PO 4 pH 5.00 buffer solution: 5.0 g of the salt were dissolved in 500 mL of pure water.The pH was adjusted with NaOH 0.2 mol L -1 , prepared by dilution from that of 2.0 mol L -1 , to pH = 5.00  0.05.
Standard solutions for the calibration curve, HPLC: 507.6 mg of the drug (containing 500.0mg of metformin hydrochloride) was exactly weighed and transferred to a 100.0 mL volumetric flask and dissolved with 50.0 mL of methanol.The volume was completed with the pH 5.00 buffer solution.From this 5000 ppm solution 8.00 mL were taken and transferred to a 200.0 mL volumetric balloon.The volume was completed with the mobile phase above described.From this 200.0ppm stock solution 120.0, 130.0, 140.0, 150.0, 160.0, and 170.0 ppm concentrations were obtained through adequate dilutions.

Samples treatment
Proposed method 20 tablets were accurately weighed in an analytical balance to 0.1 mg.From the total mass the mean weight of a tablet was calculated.An aliquot of about 1.0 g (about 800 mg of metformin hydrochloride) with accuracy of 0.1 mg was weighed into an essay tube.2.0 mL of water were added following stirring to dissolve the drug and centrifugation during 5 minutes at 4500 rpm to separate no soluble excipients.The supernatant was carefully transferred to a 10.0 mL volumetric flask.2.0 mL more were added to the residue into the tube and the process was repeated.Three extraction were performed and always the supernatant was introduced into the 10.0 mL balloon.The volume was completed to the mark with water.
HPLC method [28] 20 tablets were accurately weighed in an analytical balance to 0.1 mg and carefully crushed in a mortar.
An aliquot of the pharmaceutical preparation, containing c.a. 500.0mg of metformin hydrochloride was exactly weighed and transferred to a 100.0 mL volumetric flask and dissolved with 50.0 mL of methanol.The volume was completed with the pH 5.00 buffer solution.
From this 5000 ppm solution 2.00 mL were taken and transferred to a 50.0 mL volumetric balloon.The volume was completed with the mobile phase above described.From this 200 ppm solution a 140 ppm solution was prepared by dilution, i.e., 3.50 mL were diluted to 5.00 mL with water.[8] 20 tablets were carefully weighed to 0.1 mg and crushed in a mortar.An aliquot containing about 60 of metformin was weighed to 0.1 mg, transferred to a 150 mL beaker and solubilized with 50 mL of acetic anhydride and 4 mL of formic acid.Titration was point-to-point performed with a standard solution of perchloric acid 0.1 mol L -1 .

Proposed microgravimetric method
The proposed method was based on the reaction of metformin with nickel(II) in aqueous medium forming an insoluble complex [12].The reaction was performed inside a plastic 10 mL syringe.The 2.00 mL aliquot of the sample was first measured with a pipette, placed in a 5 mL beaker and then carefully sucked with the syringe as can be saw in Figure 2. In sequence the other solutions are successively placed in this same beaker and sucked into the same syringe.The solutions were always introduced in the following order.2.00 mL sample; 1.5 mL nickel(II) 1.0 mol L -1 ; 3.0 mL NH 3 concentrated (17 mol L -1 ); 2.0 mL NaOH 2.0 mol L -1 .The precipitate is quantitatively formed in about 3 minutes (Fig. 3).The precipitate was separated using a tube with a sintered G3 glass filter [35] (Fig. 4A) or a syringe filter (Millipore, Millex HV, 0.45 m pore, 33 mm diameter) (figure 4B) previously weighed.
In the case of the glass tube vacuum pump was used to filtrate the solid but a centrifuge can be used [35].
The solid in the filter was carefully washed with about 2.5 mL of concentrated NH 3 following drying in an oven at 120 o C for one hour.From the obtained weight of metformin as precipitate its quantity in the pharmaceutical preparation was calculated.Potentiometric method [8,9] The method recommended by the American and Japanese pharmacopoeias was used [8,9], using perchloric acid solution as titrant.

Statistical treatment
For statistical comparison of the data obtained with the proposed method versus the reference methods the t test of Student and the Snedecor F test were used [36].

RESULTS AND DISCUSSION
The orange color precipitate of nickel-metformin complex is easily formed but the solution must be strictly mixed in the indicated order: metformin, nickel, ammonium hydroxide, sodium hydroxide.Mainly in the case of sodium hydroxide solution that is added in order to increase the pH, if it is mixed before the ammonium hydroxide, nickel hydroxide precipitates and the formation of the desired complex does not occur.
It is important to pay attention to the molar excess of nickel, with respect to metformin (MF), in order to guarantee the quantitative formation of the complex.
The solution of metformin presents no color, unless an organic colorant is present.The addition of the green solution of Ni(II) turns all solution in this color.The addition of the ammonium hydroxide causes change to a blue color as the result of the Ni(NH 3 ) 6 2+ hexamine complex.With the posterior sodium hydroxide solution an orange species is formed in solution, precipitating soon after as Ni(MF) 2 .The sodium hydroxide solution must be slowly and in three portions so the precipitate will be gradually formed.
As the ammonia complexes with Ni(II), forming the hexamine complex a soluble species, the precipitation of nickel as hydroxide when sodium hydroxide is added is avoided and he excess of this cation can be easily removed by washing the precipitate with the concentrated ammonium hydroxide.
The remainder ammonia in the precipitate can be easily eliminated by heating in an oven at 120 o C.
The reaction of formation of the Ni(MF) 2 complex, which precipitates as an orange color solid, can be summarized by the following equation which clearly shows the necessity of a base to shift reaction to the right: A glass tube containing a G3 porosity sintered glass disc 35 was used to filter the precipitate or alternatively a Millipore syringe filter (Millipore, Millex HV, 0.45 m pore, 33 mm diameter).The same samples were analized using the titrimetric potenciometric procedure recommended by pharmacopeias [8,9] and by a HPLC method [28].This last procedure was preferred as it uses external standard.All the essays were done in triplicate.
Table 1.Comparison of the determination of metformin hydrochloride in pharmaceutical preparations using the proposed method, HPLC [28] and that of potentiometric titration according pharmacopoeias [8,9] It can be observed that the calculated t and F values are smaller than those tabulated, when the proposed gravimetric procedures are compared with the potenciometric official method, indicating equivalent results and precision.In the HPLC cases two small differences are observed in t values and one in F values when comparison is done with the gravimetric method where a glass tube with sintered disc is used and with the official titrimetric procedure.However, if the 99% confidence level is considered all results become is agreement.

CONCLUSION
The proposed method presents some interesting characteristics.It easy and relatively fast to be performed; it is very reliable and inexpensive; a calibration plot is unnecessary because it is an absolute method; it is a green procedure as does not uses organic or solvents or toxic substances (excepting small quantities of ammonia) and those used are in small quantities.Therefore the proposed method can be suggested for the analysis of metformin in the bulk drug and in pharmaceutical preparations.

Table 2 ) . Table 2 .
[36]lues are in milligrams per tablet  SD.Comparison of the analytical results using the Student's paired t test and the Snedecor F test[36].=(n A + n B -2) = 4; at the 95 % confidence level.t tab =2.8 and F tab =19.0; at the 99% confidence level t tab = 4.604 and F tab =99.0 Grav.t = proposed gravimetric; filtration using glass tubes with sintered disc.Grav.f = proposed gravimetric; filtration using syringe filters.HPLC = High pressure liquid chromatography.Official method: potentiometric titration.