Development and Validation of a Simple and Sensitive LC-MS/MS Method for Quantification of Metformin in Dried Blood Spot Its Application as an Indicator for Medication Adherence

Figures & data

Figure 1 Product ion spectra of [M + H]+ of metformin, afatinib (IS), fragmentation ion scans. Y-axis is Relative intensity (cps); X-axis is mass-to-charge (m/z, Da).

Figure 2 Mass chromatograms of blank dried blood spot (A), dried blood spot spiked with a metformin (8 µg/mL) (B) and afatinib (5µg/mL) (C) and dried blood spot from patient after 4 h after administration of 500 mg metformin (D).

Table 1 Summary of Back Calculated of the Calibration Curve for the Determination of MET

Nominal Concentration (ng/mL)		Mean Concentration Found (ng/mL)	Accuracy (%RSD)	%Mean Recovery ± SD
Interday statistics N=16	8	7.84 ± 0.32	4.11	98.24 ± 3.58
	24	23.65 ± 0.62	2.64	98.56 ± 4.37
	40	40.41 ± 2.36	2.36	101.05 ± 4.66
Intraday (on day 3), n = 6	8	7.8 ± 0.40	5.19	100.63 ± 4.22
	24	23.44 ± 0.75	3.2	99.17 ±3.93
	40	40.50 ± 1.23	3.05	101.5 ± 3.84

Table 2 Extraction Recovery (ER) and Matrix Effect (ME) of QC Sample of Metformin in Spiked DBS Using LC-MS/MS

QC Concentration (ng/mL)	%ER ± SD
8	98.65 ± 5.34
24	94.21 ± 3.74
40	95.56 ± 4.52

Figure 3 Stability of metformin in autosampler vials at different temperature conditions.

Table 3 Metformin Concentrations from Six Volunteers

N	Sex	Administered Drug	Time After Oral Intake (h)	Concentration (µg/mL) ± (SD)
1	F	500 mg bid	4	8.09 ± 0.53
2	F	500 mg bid	10	4.18 ± 0.55
3	M	1000 mg bid	2	15.29 ± 0.17
4	M	1000 mg bid	1	3.11 ± 0.39
5	F	500 mg bid	10	5.22 ± 0.41
6	F	500 mg bid	12	3.41 ± 0.69