Introduction

Metformin belongs to a class of drugs known as biguanides and contains the active ingredient metformin hydrochloride. Metformin oral tablets are used, in combination with diet and exercise, to treat high blood

glucose levels in patients with type 2 diabetes mellitus. Tablets are often used as monotherapy, or in combination with other anti-diabetic agents or insulin.1

This application brief describes the use of a Quasar C18 column for the analysis of metformin hydrochloride RS (Figure 1) in accordance with Dissolution Test 3 in the United States Pharmacopoeia (USP) monograph for metformin hydrochloride tablets.2

HPLC Analysis of Metformin Hydrochloride RS Using a Quasar C18 Column in Accordance with the United States Pharmacopoeia

A P P L I C A T I O N B R I E F

Liquid Chromatography

Author:

Kathryn Lawson-Wood

PerkinElmer, Inc. Seer Green, UK

2

Figure 1. Chemical structure of metformin hydrochloride.

Figure 2. HPLC analysis of metformin hydrochloride using a Quasar C18 column, 250 x 4.6 mm, 5 µm.

Experimental Conditions

Method ParametersAll HPLC method parameters are shown in Table 1.

Instrument PerkinElmer Flexar™ with PDA Plus™ Detector

Quasar C18 250 mm 4.6 mm 5 µm N9308801

Mobile Phase5:95, ACN: Buffer (5.75 mM monobasic sodium phosphate, 9.06 mM 1-pentanesulfonic acid sodium salt, pH 3.00)

Standard Medium pH 6.8 phosphate buffer

Flow Rate 1.0 mL/min

Temp 30 oC

Wavelength 230 nm

Injection Volume 40 µL

Analyte Metformin hydrochloride

Table 1. HPLC method parameters.

Solvents, Reagents and SamplesAll solvents and reagents used were HPLC grade. Phosphate buffer medium (pH 6.8, 20 mM) was prepared using potassium dihydrogen phosphate (KH2PO4) and dibasic potassium phosphate (K2HPO4).

A stock solution of USP Metformin Hydrochloride RS (0.25 mg/mL) was prepared in the phosphate buffer medium. From this solution, a working standard solution was prepared (0.05 mg/mL) in the medium. The standard solution was filtered using a 0.45 µm nylon filter (P/N: 02542880) prior to analysis.

The mobile phase buffer solution was prepared by dissolving 1.38 g of monobasic sodium phosphate in approximately 1800 mL of distilled water. To this solution, 3.484 g of 1-pentanesulfonic acid sodium salt (ion-pairing reagent) was added and dissolved. The solution was then adjusted to a pH of 3.00, using dilute phosphoric acid (5.9 %), and diluted to a volume of 2000 mL with distilled water.

Results

The analysis of metformin hydrochloride using a Quasar C18 (250 x 4.6 mm, 5 µm) column (P/N: N9308801) yields a retention of just under 14 minutes (Figure 2). Ideally suited to the analysis of small molecules, the Quasar C18 phase provides superb efficiency (over 25 % more than is required by the USP) due to the optimal bonding technology and ultra-high purity silica base. Excellent peak shape is also obtained (Table 2). The asymmetry was measured at 10 % peak height, as per USP requirements. Additionally, the peak asymmetry was measured at 5 % peak height. In both cases the asymmetry values were 1.044.

USP requires the metformin hydrochloride standard solution to have the following suitability requirements:

• Tailing factor/asymmetry: ≤ 2.0

• Column efficiency: ≥ 1500 theoretical plates

• Relative standard deviation (RSD): ≤ 2.0 %

USP also states that the RSD should be calculated, unless otherwise specified in the individual monograph, using data from five replicate injections of the analyte, if the requirement is ≤2.0 %. Data from six replicate injections are used if the RSD requirement is >2.0 %.3 As shown in Table 3, the USP precision requirements are met with an excellent level of repeatability (RSD below 0.5 %).

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Figure 3. Overlaid chromatograms of five replicate injections of metformin hydrochloride.

Table 2. Results summary.

Metformin Hydrochloride Retention time (min) Asymmetry* Column Efficiency (Plates) Peak Area (µV*sec)

Quasar Results 13.31 1.044 18989 4056631

USP Requirements n/a < 2.0 ≥ 1500 n/a

AnalytePeak Area RSD (%)

Quasar Result USP Requirement

Metformin Hydrochloride 0.12 < 2.0

Table 3. Relative standard deviation calculated using data from five replicate injections.

Conclusion

• The Quasar C18 HPLC phase offers repeatable separation of metformin hydrochloride, with the RSD below 0.5 %, meeting USP requirements of ≤ 2.0 %.

• Column efficiency and asymmetry values fall well within the USP requirements.

• The ultra-high purity silica base and low residual silanol activity yields excellent peak shape.

• Run time could be reduced by using a shorter Quasar C18 column.

References

1. R. Song, Mechanism of Metformin: A Tale of Two Sites, Diabetes Care, 39, 2016, 187-189.

2. Metformin, USP 41-NF36, United States Pharmacopoeia, 2615 – 2616.

3. Chromatography General Chapter <621>, First supplement to USP 40-NF 35, United States Pharmacopoeia.

Consumables

PhaseLength (mm)

I.D. (mm)

µmPart

Number

Quasar C18 300 3.9 5 N9308800

Quasar C18 250 4.6 5 N9308801

Quasar C18 150 4.6 5 N9308802

Quasar C18 100 4.6 5 N9308803

Quasar C18 50 4.6 5 N9308804

Quasar C18 150 4.6 3 N9308805

Quasar C18 100 4.6 3 N9308806

Quasar C18 50 4.6 3 N9308807

Quasar C18 150 3.0 3 N9308808

Quasar C18 100 3.0 3 N9308809

Quasar C18 50 3.0 3 N9308810

Quasar C18 150 2.1 3 N9308811

Quasar C18 100 2.1 3 N9308812

Quasar C18 50 2.1 3 N9308813

Quasar C18 100 4.6 1.7 N9308814

Quasar C18 50 4.6 1.7 N9308815

Quasar C18 100 3.0 1.7 N9308816

Quasar C18 50 3.0 1.7 N9308817

Quasar C18 100 2.1 1.7 N9308818

Quasar C18 50 2.1 1.7 N9308819

Quasar C18 Guard Cartridge (3/pack) 10 3 5 N9308980

Quasar C18 Guard Cartridge (3/pack) 10 3 3 N9308981

Quasar Guard Cartridge Holder - - - N9306876

Quasar C18 Method Validation Kit, 250 x 4.6 mm

(3 columns each from a different batch)

250 4.6 5 N9300940

Quasar C18 Method Validation Kit, 150 x 4.6 mm

(3 columns each from a different batch)

150 4.6 5 N9300941

*Asymmetry measured at 10 % peak height.