Methyclothiazide. Methyldopa. Metoprolol. Nitrazepam. Nortrlptyline. Oxazepam. Paracetamol. Phenylbutazone. Phenytoin. Pindolol. Prazosin. Prochiorperazine.
CLIN. CHEM. 27/3, 434-436(1981)
Rapid Liquid-Chromatographic Determination of Metformin in Plasma and Urine Bruce G. Charles,1 Noel W. Jacobsen,2 and Peter J. Ravenscroft’ We describe an analysis for metformin in plasma and urine by use of cation-exchange “high-performance” liquid chromatography. The assay requires only 0.5 mL of sample and involves pretreatment with trichioroacetic acid containing an internal standard (1-propylbiguanide), followed by centrifugation and injection into the chromatograph. The column eluent is monitored at 230 nm. Metformin and 1-propylbiguanide give retention times of 8 and 10 mm, respectively. Within-day and between-day precision (CV) for the assay of plasma containing 2mg (1.2 X iO mol) of metformin hydrochloride per liter was 3.6 and 3.9%, respectively. Similar precision was obtained for urine. The limit of detection is about 0.1 mg (6.0 X 10 mol) of metformin hydrochloride per liter. Several commonly used drugs, including other oral hypoglycemic agents, do not interfere with this method. AddItional diabetes
Keyphrases: -
monitoring
therapy
drug assay
Metformin (1,1-dimethylbiguanide) is frequently used to treat diabetes mellitus, both in obese patients whose diabetes is uncontrolled by diet and in patients who fail to respond to therapy with sulfonylurea (1). High concentrations of biguanides in plasma have been associated with an increased incidence of lactic acidosis, particularly in patients in acute renal failure (2-4). The development of methods for the measurement of metformin concentrations in biological fluids has received little attention. This may be due, in part, to the high polarity of metformin, which precludes efficient extraction of the molecule from biological fluids (6). Moreover, the polarity of metformin complicates analysis for the compound by gas-liquid chromatography and necessitates considerable sample work-up to prepare the s-triazine derivatives (6-8). A similar procedure has been adopted for the pretreatment of urinary samples of metformin for subsequent analysis by liquid chromatography (9). Here we describe a rapid, sensitive, and precise method for determination of metformin in plasma and urine by cationexchange “high-performance” liquid chromatography (HPLC). Our method requires only 0.5 mL of sample, involves minimum sample work-up, and avoids the need to derivatize metformin before chromatography.
(Rheodyne Inc., Berkeley, CA 94710) and a Waters Model 450 variable-wavelength detector. A 25 cm X 4.6 mm Partisil-lO SCX cation-exchange column was used (Reeve-Angel, Whatman, Inc., Clifton, NJ 07014). Reagents and Standards A stock solution of metformin
hydrochloride (S.N.E.L. Aron, Suresnes 92150, France) was prepared by dissolving 100 mg in 100 mL of distilled water. Working standards, obtained by diluting the stock solution in drug-free plasma and urine, were routinely prepared to contain 10, 5,2, 1,0.5, and 0.2mg of metformin hydrochloride per liter.
1-Propylbiguanide sulfate, the internal standard, was by the following procedure. Propylamine (9 g, 101 mmol), cyanoguanidine (7.5 g, 60 mmol), and copper sulfate pentahydrate (11.6 g, 31 mmol) were mixed together in 75 mL of water and heated in a sealed tube for 12 h at 100 #{176}C. The reaction vessel was cooled, opened, and the contents were diluted with 350 mL of water. The solution was re-heated to 80#{176}C and the copper salts were precipitated from the solution by passing a stream of H2S through the hot solution. The copper sulfide was filtered off and the filtrate evaporated under reduced pressure at 100 #{176}C. The residual solid was recrystallized from hot ethanol and the product (3.7 g, 16% yield) synthesized
dried at 100 #{176}C to yield 1-propylbiguanide, mp 194-196 #{176}C LFound: C, 31.0; H, 7.4; N, 36.4; S, 8.6%. Calculated for (C5H13N5)2H2S04: C, 31.2; H, 7.3; N, 36.4; S, 8.3%]. A stock solution of the internal standard was prepared by dissolving 30mg of 1-propylbiguanide sulfate in 10 mL of distilled water. This solution was stable for several weeks at 4 #{176}C. A working internal standard solution was prepared weekly by diluting 0.1 mL of the stock solution to 10 mL with an aqueous solution of trichloroacetic acid (1.2 mmoIfL). All other chemicals were
()
(8)
Materials and Methods Apparatus Our apparatus for HPLC consisted solvent-delivery
of a Model M6000A system (Waters Associates, Inc., Milford, MA
27486) connected to a Model 7120 six-port sample injector Department
of
Clinical
Hospital, Brisbane Q, Australia
Pharmacology,
Princess
4102.
Alexandra
#{176}Department of Organic Chemistry, University of Queensland, St. Lucia Q, Australia 4067. Received Nov. 11, 1980; accepted Dec. 16, 1980.
434 CLINICALCHEMISTRY,Vol.
27, No. 3, 1981
0’4
812 MIII
04812 MIII
Fig. 1. Chromatograms supplemented with 2 mg 6 tg of 1-propylbiguanide;
04812 MIII
04812 MIII
04812 MIII
for (A) blankplasma;(B) blank plasma of metformin hydrochloride per liter and
(C) patient’s plasma containIng 2 mg of metformin hydrochloride per liter; (C) blank urine; (E) patient’s urine containing 3 mg of metformin hydrochloride per liter.
UrIne samples were dIluted 200-fold with distilled water before assay. Peaks are: A, metformin; B, 1-propylbiguanide(internal standard)
8
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METFORI’IIN CONCENTRATION (MG/LITRE) Fig.2. Standardcurve:ratio ofpeak heit ofmetformin topeak heightof 1-propylbiguanide vs metformin concentrationIn plasma (#{149}) and urine(0)
0
.IIs
j LL I-
I
400
#{149}II
$1’.
Data poInt represents the mean of 10 replIcate determinations
0 of analytical
8
I.
or
0
grade.
Procedure
PLASMA
Plasma. Plasma (0.5 mL) was pipetted into11 X 100 mm Pyrex glass tubes and 0.2 mL of the working internal standard solution was added to each tube. The contents of the tubes were vortex-mixed for 5 s and allowed to stand for about 10
mm, then transferred to polyethylene micro test-tubes (5.5 X 46 mm, 0.5 mL) and centrifuged at approximately 5000 X g for 5 mm (Microfuge B; Beckman Instruments, Inc., Palo Alto, CA 94304). A 100-giL portion of the clear supernatant fluid was injected into the liquid chromatograph. Urine. The procedure for urine samples was the same except that the working internal standard solution was prepared in distified water. When necessary, urine samples werediluted in distilled water before HPLC. A 200-fold dilution was suitable in most cases. The mobile phase consisted of ammonium dihydrogen phosphate solution (0.03 mol/L) adjusted to pH 2.4 with orthophosphoric acid, and was delivered at 3 mL/min. The temperature of the column was maintained at 50#{176}C, in a water bath. The column eluent was monitored at 230 nm and chromatograms were obtained with a pen recorder having a 1-mV and 10-mV range and operatedat a chart speed of 0.25 cm! mm.
URINE
Fig.3. Plasma (#{149}) andurine(0) concentrationsof metformin in patients, as determined
by HPLC
Results and Discussion To minimize variation generatedby sample clean-upproceduresand changes in theinjection volume,it was necessary to use an internalstandardin the assay. 1-Propylbiguanide was chosen as a suitable internal standard becauseit is neither a drug nora metabolite ofmetformin, but hassimilar spectral properties to metformin and has a comparableretention time on the cation-exchangecolumn. Metformin had a retentiontime of 8 mm and 1-propylbiguanidea retention time of 10 mm under the chromatographic conditionsspecified. Figure 1 shows representative chromatograms ofblankplasma and urine and samples containing metformin hydrochloride. In the case of both plasma and urine,metformin and 1-propylbiguanide were wellresolved from peakscausedby endogenousconstituents ofthesamples. Calibration graphs were routinely constructedby assaying a rangeofmetformin hydrochloride concentrations from 0.2to
Table 1. Precision of HPLC Assay of Metformin in Plasma and Urine8 Concn psas.nt, mg/L
Between-day(n= Mean SD CV, % Within-day
Mean SD CV, % II
10
Plaema 2
UrIn 0.2
10
2
0.2
10)
3.48 0.107
0.70 0.027
3.1
3.9
3.47 0.114 3.3
0.68
0.082 0.0093 11.3
3.42
0.025
0.060 0.0040
3.6
6.6
3.62 0.029 0.8
0.102
3.0
0.69 0.023 3.3
0.092 0.0083
9.1
(n = 10)
0.76
0.069
0.0098
0.0053
1.3
7.6
Results are expressed as the peak-height ratios of metformin to 1-propylbiguanide, the internal standard.
CLINICAL CHEMISTRY,
Vol. 27, No.
3, 1981 435
Table 2. Medication Being Received by Patients Whose Plasma and Urine Were Analyzed for Metformin Allopurinol
Ampicillin
Methyclothiazide Methyldopa Metoprolol Nitrazepam
Amylobarbital
Nortrlptyline
Aspirin
Oxazepam Paracetamol Phenylbutazone
Amiloride
Amitriptyline
Caffeine Carbamazepine Chloropropamide Chlorothiazide Clonidine Danthron Dextropropoxyphene Diazepam
Digoxin Dioctylsodium
sulfosuccinate Doxepin Ephedrine
Ethylestranol Furosemide Glibenclamide
Glyceryltrinitrate Hydrochiorothiazide
Insulin
Phenytoin Pindolol Prazosin
by assaying theabove concentrations of metformin hydrochloride in plasma and urine10 times on the same day.To measure the between-day precision, we assayed one plasma sample of metformin and one urinarysample daily on each of 10 days usinga new internal standard solution each sion was determined
day.
We assessedtheclinical performanceoftheassayby measuringmetforminconcentrations in plasma and untimed urine samplesfrom patientsattending a diabeticclinic (Figure 3). Most ofthepatients studiedwerealsobeingtreated with other drugs, includingantihypertensives, diuretics, antimicrobials, analgesics, and varioussulfonylureas (Table 2).The peaks corresponding tometforminand 1-propylbiguanide were well resolved from peaksascribable tothepresence of any of these drugsortheirmetabolitesinplasma and urine. We fmd that atleast 30 metformin assayscan be performed by one personduringa normal working day.
Prochiorperazine
Propranolol Quinine Salbutamol
Sodium chromoglycate Sorbidenitrate Sulfamethoxazole Theophylline Thyroxine Tolbutamide Trimethoprim
Verapamil Vitamin B complex Vitamin mixture Warfarin
10 mg/L (1.2X 106 to6.0X i0 molfL) inplasma and urine and plottingthe peak-height ratio of metformin to 1-propylbiguanideagainst metformin concentration. The mean for10 such calibration graphs for metformin in plasma and urine are presented in Figure 2. These plots were linear (r >0.99) over the 0.2 to 10 mg/L range and passed through the origin. Furthermore, the calibration graphs for metformin in plasma and urine were almost superimposable, suggesting that, ifnecessary, the graph for plasma metformin could also be used for measuring urinary concentrations of metformin, and vice versa. The limit of detection of metformin hydrochloride was found to be about 0.05 to 0.1 mgfL (3.0 X 10 to 6.0 X iO mol/L).
To determinethe precision oftheassay(Table1),we supplemented metformin-free plasma and urine withmetformin hydrochloride to give concentrations of 0.2, 2, and 10 mg!L, a range of concentrations thatencompasses thosemeasured clinically (10, 11). The samples were subdivided and stored at -20 #{176}C until assayed. Metformin was stable under these storage conditions for at least four weeks. Within-day preci-
436 CLINICALCHEMISTRY,Vol.27,No.3,1981
Inconclusion, our method issimpletoperform and provides a specific, rapid,and precisemeans ofmeasuringmetformin concentrations in plasma and urine.The assayiscurrently being used in our department to measure serum metformin concentrations, effect, and toxicity indiabeticpatients.
References 1. Clarke, B. F., and Duncan, L. J. P., Biguanide treatment in the management of insulin independent (maturity onset) diabetes: Clinical experience with metformin. Res. Clin. Forums 1, 53 (1979). 2. Assan, R., Heuclin, Ch., Ganeval, D., eta!., Metformin-induced lactic acidosis in the presence of acute renal failure. Diabetologia 13, 211 (1977). 3. Conlay, L.A., Karim, J. H., Martin, S.B., and Loewenstein, J. E., Serum phenformin concentrations in patients with lactic acidosis. Diabetes 26,628 (1977). 4. Luft, D., Schmulling, R. M., and Eggstein, M., Lactic acidosis in biguanide treated diabetes. Diabetologia 14,75 (1978). 5. Casey, C., Connor, H., Phillips, P., et al., Metformin: Absorption and disposition in healthy subjects and in diabetic patients. Br. J. Clin. Pharmacol. 8, 382P (1979). 6. Lennard, M. S., Casey,C., Tucker, G. T., and Woods, H. F., Determination of metformin in biological samples. Br. I. Clin. Pharmacof. 6, 183 (1978). 7. Matin, S. B., Karam, J. H., and Forsham, P. H., Simple electron capture gas-chromatographic method forthedetermination oforal hypoglycemic biguanides inbiological fluids. Anal. Chem. 47, 545 (1975).
S. Brohon, J., and Noel, M., Determination of metformin in plasma at therapeutic levels by gas-liquid chromatography using a nitrogen detector. J. Chromatogr. 146, 148 (1978). 9. Ross, M. S. F., Determination ofmetformin inbiological fluids by derivatization followed by high performance liquid chromatography. J. Chromatogr. 133,408(1977). 10. Noel, M., Kinetic study of normal and sustained release dosage forms of metformin in normal subject. Re8. Clin. Forums 1, 35 (1979).
11. Pentikainen, P. J., Neuvonen, P. J., and Penttila, A., Pharmacokinetics of metformin after intravenous and oral administration to man. Eur. J. Clin. Pharmacol. 16, 195 (1979).
