June 2023

Medical Mass Spectrometry Vol. 7 No. 1

Research Paper

Simultaneous quantiﬁcation of multiple anti-diabetic drugs inwhole blood by ultra-performance liquid chromatography-tandem
mass spectrometry
1,2
2,3
2,3
Takayoshi Suzuki *, Tadashi Ogawa , Masae Iwai ,
2,3

2,4

2,3

Tomohito Matsuo , Fumio Kondo , Hiroshi Seno

Department of Medical Technology, Shubun University, 6 Nikko-cho, Ichinomiya, Aichi 491‒0938, Japan2
Department of Legal Medicine, Aichi Medical University School of Medicine,1‒1 Yazakokarimata, Nagakute, Aichi 480‒1195, Japan
3
Poison Analysis Center, Aichi Medical University School of Medicine, 1‒1 Yazakokarimata, Nagakute, Aichi 480‒1195, Japan
4
Department of Biomedical Sciences, Chubu University College of Life and Health Sciences,
1200 Matsumoto-cho, Kasugai 487‒8501, Japan
1

Abstract

An analytical method was developed and validated for the simultaneous quantiﬁcation of multiple anti-diabetic

drugs in whole blood using ultra-performance liquid chromatography-tandem mass spectrometry. Chromatographic separation was achieved on a CAPCELL PAK INERT ADME-HR column. The mobile phases consisted of water containing
0.1% formic acid and acetonitrile containing 0.1% formic acid. Quantiﬁcation of 10 antidiabetic drugs was performed by
selected reaction monitoring with each product ion referenced against 4 isotopically labelled internal standards. Calibration
curves exhibited good linear relationships ranging from 0.0002‒20μg/mL, with correlation coeﬃcients exceeding 0.996.
The limits of detection were estimated to be 0.0001‒0.005μg/mL. The intra- and inter-day accuracies and precisions were98.6‒105.5% and 0.1‒8.0%, respectively. The recovery eﬃciencies were in the range of 74.4‒102.3%. Matrix eﬀects wereobserved ranging from −29.1 to 534.6%. Stability tests of the analytes revealed that gliclazide was relatively unstable
when stored long-term (28 days) at −30°
C. We expect that the method described in the present study will be useful for various studies such as clinical and forensic toxicological investigations.
Key words: anti-diabetic drugs, whole blood, ultra-performance liquid chromatography-tandem mass spectrometry, quantiﬁcation, forensic toxicology

Introduction(around 90‒95%). It is considered a chronic metabolic syn2)

Increasing numbers of patients are being diagnosed withdrome . It includes individuals with insulin resistance anddiabetes mellitus, constituting a serious health problem .
Diabetes mellitus type 2, which is non-insulin dependent

1)

relative insulin deﬁciency. The primary drug therapies fordiabetes, accounts for the majority of diabetic patientsbiguanides, insulin sensitizers, alpha glucosidase inhibitors,
incretin mimetics, amylin antagonists and sodium-glucose

diabetes mellitus type 2 consist of insulin secretagogues,* Corresponding author

co-transporter-2 inhibitors . Among them sulfonylureas andbiguanides can cause life-threatening hypoglycemia and/or
3)

Takayoshi Suzuki
Department of Medical Technology, Shubun University,
6 Nikko-cho, Ichinomiya, Aichi 491‒0938, Japan

4,5)

lactic acidosis.

The serum glucose level is regulated within a narrow

Tel: ＋81‒586‒45‒2101, Fax: ＋81‒586‒45‒5410
E-mail: suzuki.ta@shubun.ac.jp

range by multiple factors under physiological conditions,

Received: September 1, 2022. Accepted: November 9, 2022.

but is greatly modiﬁed in the death process and after

Epub January 13, 2023.

death . Therefore, it is not adequate to determine the blood
glucose level to diagnose the life-threating hypoglycemia

6)

DOI: 10.24508/mms.2023.06.002
35

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

after death. The risk of developing hypoglycemia is also
increased in case of renal impairment, reduced food con-mass spectrometry (UPLC-MS/MS). In this study, we
aimed to develop a method for the simultaneous quantiﬁca-

sumption or with simultaneous intake of drugs whichtion of ﬁve sulfonylureas (acetohexamide, chlorpropamide,
gliclazide, glibenclamide and glimepiride) and two bigua-

7)

potentiate the action of sulfonylureasʼ (i.e., salicylates) .
Furthermore, the measurement of lactate after death is diﬃ-

nides (buformin and metformin) together with three othercult since it has been shown that lactate concentrations in

drugs (pioglitazone, alogliptine and vildagliptin) used inblood and other matrices still increase for some time after

combination. The analysis focused on whole blood samples
using UPLC-MS/MS.

8)

death as a result from anaerobic glycolysis . Therefore, it isimportant to analyze such drugs in biological ﬂuids as aMaterials and Methods

part of forensic toxicological examination.

Chemicals and materials

In forensic toxicology, almost all postmortem analyses
9,10)

. To

Acetohexamide was purchased from MP Biochemicals

our knowledge, there are no reports of the simultaneous(Santa Ana, CA, USA). Glibenclamide and metforminhydrochloride were obtained from Sigma-Aldrich (St.

are performed on whole blood because of hemolysis

quantiﬁcation of sulfonylureas and biguanides in whole
blood by ultra-performance liquid chromatography-tandem

Fig. 1.

Louis, Mo, USA). Buformin hydrochloride, chlorpropa-

Chemical structures of the analytes and internal standards.
36June 2023

Medical Mass Spectrometry Vol. 7 No. 1

mide, gliclazide, glimepiride and pioglitazone were pur-chemicals used in this study were of the highest purity com-chased from Fujiﬁlm Wako Pure Chemical (Osaka, Japan).

mercially available. Laboratory distilled water was puriﬁedusing a Direct-Q UV 3 (Millipore, Molsheim, France).

Alogliptin was obtained from Bio Vision Inc. (Milpitas,
CA, USA). Vildagliptin and metformin-d6 hydrochloride

Human whole blood was obtained from Tennessee Bloodcame from Cayman Chemical (Ann Arbor, MI, USA).
Vildagliptin-d3 and pioglirazone-d4 were purchased from

Services (Tennessee, USA).

Toronto Research Chemicals, Inc. (Ontario, Canada). Tolubutamide-d9 was obtained from Santa Cruz Biotechnology

Sample preparation procedure
A 100μL whole blood sample was mixed with 10μL IS
solution (1μg/mL of metformin-d6 and tolubutamide-d9 and(Dallas, TX, USA). The chemical structures of the analytes

0.1μg/mL of vildagliptin-d3 and pioglirazone-d4), 100μL

are shown in Fig. 1. Stock solutions of all substances wereprepared at concentrations of 0.4‒1 mg/mL in methanol andwere stored at −80°
C until analysis. The IS stock solutions

methanol, and 200μL acetonitrile. The mixture was vortexedfor 60 s and centrifuged at 15,000 g for 10 min. The superna-

were prepared in methanol to give a concentration of
100μg/mL and were stored at −80°
C. LC-MS grade aceto-tant was transferred to another tube, and evaporated with acentrifugal evaporator (CVE-2000; Tokyo Rikakikai, Tokyo,

nitrile, methanol and formic acid were purchased from Fuji-

Japan). The residue was reconstituted in 100μL of 0.1%
formic acid, and used for the analysis by UPLC-MS/MS.

ﬁlm Wako Pure Chemicals (Osaka, Japan). Other common
Table 1.
Analyte

SRM transitions and parameters for detection of the analytes and IS

Molecular weight

Retention time (min)SRM transition (m/z)

Cone voltage (V)Collision energy (eV)

Metformin129.16

1.36

130

→

71*

28

22

Buformin157.22

2.23

158

→

60

28

14

85*

26

20

60

26

14

130
158
Alogliptin

339.39

2.23

340
340

Vildagliptin

303.402.24

304

Pioglitazone

356.44

2.43

357

Chlorpropamide

276.74

3.36

304
357
277
277
Acetohexamide

324.40

3.53

325
325

Gliclazide

323.41

4.01

324

Glibenclamide

494.00

4.49

494

Glimepiride

490.62

4.56

491

324
494
491
Metformin-d6 (IS)135.20

1.35

136
136

Vildagliptin-d3 (IS)306.42

2.24

Pioglitazone-d4 (IS)360.46

2.42

Tolbutamide-d9 (IS)279.40

3.50

307
307
361
361
280
280

Notes: *Transitions used for quantiﬁcation of the compounds.

37

→

→

→

116*

38

30

→

89
154*

38

58

32

18

→

97

32

28

134*

44

30

→

119

44

46

175*

22

20

111

22

32

→

→

→
→
→

119*

26

28

→

91

26

44

110*

28

22

→

91

28

34

369*

24

14

→

169

24

42

352*

26

14

126

26

28

77*

28

20

94
151*

28

14

32

26

157

32

18

138*

48

28

123

48

46

91*

26

32

83

26

14

→
→

→

→

→

→

→
→

→

→

→
→

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

UPLC-MS/MS conditions

residue extracted from whole blood. The matrix eﬀects

UPLC-MS/MS analysis was run on a Waters ACQUITY

were calculated by [(Aeb/Ans −1) × 100], where Aeb and

UPLC system, which included an ACQUITY UPLC binary

Ans represent the analyte peak area of the extracted blankpump and a sample manager (Waters, Milford, MA, USA).

whole blood that was directly spiked with the analytes and

The LC column used for chromatographic separation was athe neat analyte solution, respectively. The recovery rates

CAPCELL PAK INERT ADME-HR (2.0 mm I.D. × 100 mm,

were calculated by [(Aex/Aeb) × 100], where Aex and Aeb

particle size 3μm). The column temperature was maintained

represent the analyte peak area of the extracted analytes inat 40 °
C. Gradient elution was employed using a mobile

whole blood and extracted blank whole blood directly

phase consisting of mobile phase A (water containing 0.1%

spiked with the analytes, respectively.
Stability tests, such as freeze‒thaw stability, short-term

formic acid) and mobile phase B (acetonitrile containing
0.1% formic acid) at a ﬂow rate of 0.2 mL/min (0‒1 min)stability and long-term stability were carried out at three
diﬀerent concentrations. We evaluated several storage con-and 0.3 mL/min (1‒10 min). The mobile phase systemstarted at 99% A for 1 min. A linear gradient was used toditions, including room temperature for 4 h, −30°
C andramp from 60% A to 1% A within 5 min and maintained at
1% A for 1 min. The mobile phase was then returned to−80°
C for 28 days and three freeze‒thaw cycles. Eachfreeze‒thaw cycle consisted of thawing the spiked samples

99% within 0.01 min and maintained at 99% A for 3 min toat room temperature and refreezing for a minimum of 12 hequilibrate the column for the next sample.

between each cycle.

The MS/MS was operated with an electrospray ionization source in the positive ionization mode on a tandem

Results and Discussion

quadrupole mass spectrometer (ACQUITY TQD; Waters).
Quantiﬁcation was performed by selected reaction monitor-

Optimization of analytical conditions

ing (SRM) using peak areas. The parameter settings were

The ﬁrst step in method development was the optimization of mass spectrometry parameters by infusing individ-

as follows: capillary voltage, 3.0 kV; source temperature,ual standard solutions at concentrations of 0.1‒1μg/mL.

150°
C; desolvation temperature, 450°
C; desolvation and

The product ion mass spectra of the analytes and IS wereobtained from the protonated molecular ions. Using proton-cone gas, nitrogen with ﬂow rates of 800 and 50 L/h,
respectively; and collision gas, argon with ﬂow rate of
0.15 mL/min. Table 1 shows the optimized SRM parame-

ated molecular ions as precursor ions, other MS/MS param-

ters and retention times for each compound and IS. All data

The chromatographic elution of the analytes was opti-

were acquired in the centroid mode and processed using

mized by examining several analytical columns and mobile

MassLynx NT 4.1 software with the QuanLynx programphases. We found that a CAPCELL PAK INERTADME-HR column with a mobile phase of water contain-

eters and transitions were determined as shown in Table 1.

(Waters).

ing 0.1% formic acid and acetonitrile containing 0.1% for-Method validation

mic acid provided the best chromatographic separation.

The method was validated according to the US FDA

SRM chromatograms of an extract from a whole blood

guidelines on bioanalytical method validation and related
11)
documents . The intra- and inter-day accuracies and preci-

sample spiked with 0.02μg/mL buformin, alogliptin,
vildagliptin, pioglitazone, glibenclamide and glimepiride,
0.07 μg/mL gliclazide, 0.2μg/mL metformin, chlorpropa-

sions were determined by conducting 5 experiments in theon 5 diﬀerent days, respectively. The precisions weremide and acetohexamide, 0.1μg/mL pioglitazone-d4 and1μg/mL metformin-d6, vildagliptin-d3 and tolbutaminde-d9

expressed as the coeﬃcient of variation. The accuracies andare shown in Fig. 2. The peaks of the analytes were clearly

precisions should be within 20% for the LOQ and 15% forvisible with retention times within 5 min, and there were noother concentrations.

peaks that interfered with the measurements.

course of a single day and by conducting one experiment

The matrix eﬀects and recovery eﬃciencies were deter-

Reliability of the method

mined by the following method. For matrix eﬀects, two setsof samples were prepared by directly spiking the analytes

The method was validated by characterizing a series of

into reconstitution solution with or without the presence ofparameters such as the limits of detection, linearity, accu38

June 2023

Medical Mass Spectrometry Vol. 7 No. 1

racy, precision (intra- and inter-day), recovery eﬃciencies
and matrix eﬀects. Calibration curves were prepared byadding appropriate calibrators to blank whole blood. As
shown in Table 2, calibration curves were linear in the range
from 0.005‒20μg/mL for metformin, 0.005‒5μg/mL for
buformin, 0.001‒5μg/mL for alogliptin, 0.0002‒2μg/mL for
vildagliptin, 0.0002‒5μg/mL for pioglitazone, 0.01‒20μg/mL
for chlopropamide, 0.01‒20μg/mL for acetohexamide,
0.005‒10μg/mL for gliclazide, 0.001‒3μg/mL for glibenclamide and 0.001‒3μg/mL for glimepiride, with correlation coeﬃcients exceeding 0.996. These data established thehigh degrees of linearity within these ranges. The limits ofdetection (LOD), deﬁned as the concentrations giving a signal-to noise ratio of 3 : 1, were estimated to be 0.0001‒
0.005μg/mL under the UPLC-MS/MS conditions used in thisexperiment. The sensitivities of the present results were equal
12‒19).
to or greater than those of previous reports
The intra- and inter-day accuracies and precisions were
evaluated at four levels, the results of which are summarized in Table 3. The accuracies and precisions were 98.6‒
105.5% and 0.1‒8.0%, respectively. Their values were
acceptable at all concentrations. The recovery eﬃciencies
and the matrix eﬀects of the samples are shown in Table 4.
The recovery eﬃciencies were in the range of 74.4‒
102.3%. Matrix eﬀects were observed ranging from −29.1
to 534.6%. The wide range of matrix eﬀect observed in thepresent study might come from the lack of ability of protein
Fig. 2.

precipitation using organic solvent. Further experiments

SRM chromatograms of an extract from whole
blood sample spiked with analytes and internal
standards.

aimed at reducing the amount of matrix compounds using
solid phase extraction, QuEChERS and other devices are
required.

Table 2.

Regression equations, correlation coeﬃcients (r), limits of detection (LOD) and therapeutic concentration for the
analytes

AnalyteMetformin
Buformin
Alogliptin
Vildagliptin
Pioglitazone
Chlorpropamide
Acetohexamide
Gliclazide
Glibenclamide
GlimepirideaRange(μg/mL)

Regressionaequation

Correlation coeﬃcient(r)

LOD(μg/mL)

Therapeutic concentration(μg/mL)0.005‒20
0.005‒5y＝3.081x＋0.3322

0.999

0.002

y＝1.902x＋0.0270

0.999

0.002

0.25‒1.3
0.15‒0.58

0.001‒50.0002‒2y＝189.2x＋1.503

0.999

0.0005y＝482.8x＋4.636

0.999

0.00010.0002‒50.01‒20y＝108.3x＋4.777

0.999

0.0001y＝3.551x＋0.5240

0.999

0.005

0.01‒200.005‒10

y＝2.948x＋0.2873

0.999

0.005

y＝20.17x＋4.843

0.997

0.002

0.001‒30.001‒3

y＝7.236x＋0.4765

0.996

0.0005y＝9.736x＋0.5579

0.997

0.0005

Values are the means of ﬁve replicate determinations.

39

0.025‒0.2
0.3‒0.5
0.1‒1.530‒36321‒56
0.5‒2.0
0.05‒0.2
0.09‒0.5

June 2023

Table 3.

Medical Mass Spectrometry Vol. 7 No. 1

Intra- and inter-day accuracy and precision dataof the analytes in whole blood
Inter-day
Intra-dayConcentration
added
Accuracy Precision Accuracy Precision(μg/mL)(%)(CV, %)(%)(CV, %)aAnalyteMetformin

Buformin

Alogliptin

Vildagliptin

Pioglitazone

Chlorpropamide

Acetohexamide

Gliclazide

Glibenclamide

GlimepirideaTable 4.

The recovery eﬃciencies and matrix eﬀects of theanalytes in whole blood

a

0.005

100.1

3.2

99.9

3.2

0.2

98.6

2.1

100.3

1.2

2

100.8

2.3

100.7

1.2

15

98.8

1.4

99.5

0.8

0.005

102.3

5.9

103.2

2.6

0.02

100.4

3.5

99.6

2.4

0.2

105.5

4.7

102.3

3.5

3

104.2

3.1

102.8

1.3

0.001

101.2

3.0

101.0

0.7

0.02

101.7

1.4

101.5

0.1

0.2

100.2

1.6

101.0

1.0

3

99.5

1.6

100.3

0.6

0.0002

100.1

2.0

99.9

1.2

0.02

102.1

0.8

102.5

1.5

0.2

100.9

0.8

100.7

0.6

1.5

99.7

0.3

100.2

0.4

0.0002

100.6

8.0

99.4

1.5

0.02

99.9

1.5

103.3

2.5

0.2

100.7

1.5

101.5

0.7

3

101.3

0.5

102.2

2.2

0.01

100.9

1.9

101.0

1.8

0.2

102.3

1.9

102.7

3.2

2

100.9

4.7

104.5

2.7

15

99.8

1.4

99.5

1.6

0.01

99.5

2.5

99.6

0.3

0.2

100.6

1.1

101.1

0.9

2

101.9

0.9

103.5

1.5

15

99.4

0.7

99.6

2.9

0.005

100.4

0.7

100.1

0.4

0.07

100.5

0.9

100.6

0.8

0.7

100.6

3.3

100.8

3.0

7

99.2

2.1

100.3

3.8

0.001

102.0

5.4

100.4

2.0

0.02

101.9

1.7

102.2

0.3

0.2

104.4

2.7

105.1

4.1

1.5

100.4

2.0

99.4

1.1

0.001

99.5

2.2

100.0

0.4

0.02

99.9

0.6

101.3

1.3

0.2

99.6

1.6

101.7

1.8

1.5

99.8

0.5

99.9

1.3

AnalyteMetformin

Buformin

Alogliptin

Vildagliptin

Pioglitazone

Chlorpropamide

Concentration
added(μg/mL)

Recoveryaeﬃciency(%)

Matrix
a,b
eﬀect
(%)

0.2

75.5±1.1

129.9±11.2274.4±1.3

39.7±9.11581.0±1.3

2.6±1.00.02

76.8±5.2

42.6±3.80.278.6±1.5

6.0±11.2

377.8±3.7−22.2±4.90.02

80.0±0.9

0.7±4.00.2

84.2±2.5

3.7±1.3383.0±3.5−16.8±3.6

0.02

80.7±2.2

24.6±2.2

0.2

96.4±2.3

12.9±9.91.593.4±2.3−1.0±1.3

0.02

94.5±12.4

534.6±67.5

0.2

91.5±4.5

237.6±26.5394.3±3.4

2.3±4.2

0.2

97.1±12.2

70.8±6.22

90.6±18.3

6.3±6.81592.0±11.0−6.6±4.1

0.2

94.9±14.6

44.6±6.8290.6±21.7−5.0±6.71589.3±7.9−3.3±3.8

0.07

99.6±16.1

33.9±6.4

0.7

92.8±14.7−9.6±3.0795.9±4.4−7.0±5.2

0.02

94.3±10.1

7.7±2.2

0.2

92.7±11.1−6.6±3.7

1.5

98.3±18.3

8.0±4.70.02

90.8±15.5

53.7±16.8

0.2

92.9±12.2−5.0±6.0

1.5

93.7±6.2−5.0±7.2

Metformin-d6 (IS)199.3±2.0−29.1±1.4

Vildagliptin-d3 (IS)1102.3±12.1

55.4±17.7

Pioglitazone-d4 (IS)0.1

99.0±5.4−6.8±5.3

Tolbutamide-d9 (IS)198.7±1.5

2.8±2.7

Acetohexamide

Gliclazide

Glibenclamide

GlimepirideaValues are expressed as the means±SD (n＝5).
bPositive values account for signal enhancement and negative values for signal
suppression.

The percentages of initial concentrations after storage
under diﬀerent conditions are shown in Table 5. The stabili-

Values are the means of ﬁve replicate determinations.

ties at the tested concentrations are acceptable except for
gliclazide. Gliclazide was relatively unstable when stored
long-term (28 days, −30°
C) at concentrations of 0.07 andCarry-over eﬀect was not observed when washing solvent (methanol) was injected after the analysis of high con-

0.7μg/mL. Thus, it is necessary to pay attention to stability.

centration samples. The phenomenon that a peak area gradually increased was not also observed even though theConclusions

same sample was analyzed repeatedly. Collectively, there
was no evidence of carry-over eﬀect in the analysis of the

In this study, we developed and validated a method for
simultaneous quantiﬁcation of sulfonylureas and bigua-target compounds.

nides in whole blood by UPLC-MS/MS. We expect that the40

June 2023

Table 5.

Stability of the analytes in whole blood underdiﬀerent storage conditions

AnalyteMetformin

Buformin

Alogliptin

Vildagliptin

Pioglitazone

Chlorpropamide

Acetohexamide

Gliclazide

Glibenclamide

GlimepirideaMedical Mass Spectrometry Vol. 7 No. 1

Pharmacother 131, 110708, 2020.
3) Artasensi A, Pedretti A, Vistoli G, Fumagalli L: Type 2 diabetes mellitus: A review of multi-target drugs. MoleculesLong-term,a
28 days(%)

Concentration
added(μg/mL)Freezethaw,a
3 cycles(%)

Short-term,
4 h, roomatemperature
(%)

0.2

98.3

102.5

106.0

104.6

2

98.2

101.0

102.0

99.4

15

94.7

104.3

100.7

100.4

0.02

98.6

96.5

92.4

105.2

0.2

92.1

98.1

89.8

103.9

3

93.6

98.1

91.8

100.3

0.02

99.2

97.5

99.5

102.1

0.2

99.2

99.1

97.3

97.2

6) Chen J-H, Michiue T, Inamori-Kawamoto O, Ikeda S,

3

96.0

103.2

97.6

98.7

0.02

95.6

100.2

98.8

100.6

Ishikawa T, et al: Comprehensive investigation of post-

0.2

95.8

99.6

98.6

98.6

1.5

98.3

101.4

98.8

101.0

0.02

95.4

103.1

96.3

97.9

0.2

92.9

101.4

94.3

97.5

3

92.5

101.0

92.9

97.6

0.2

103.9

107.2

101.3

103.0

2

98.6

102.4

100.5

101.2

15

101.6

104.0

109.6

109.7

0.2

96.1

105.9

96.6

96.5

2

94.0

100.8

96.0

96.4

15

98.4

102.4

103.2

102.4

0.07

96.2

100.2

68.8

98.3

0.7

94.5

99.7

53.4

95.1

7

98.2

98.9

81.8

98.7

0.02

97.7

102.3

93.3

104.0

0.2

92.6

100.5

97.0

100.1

1.5

93.8

105.8

97.2

109.6

0.02

97.3

105.2

95.6

99.6

10) Karinen R, Vindenes V, Hasvold I, Olsen KM,

0.2

93.3

106.7

95.1

108.2

1.5

93.3

102.1

97.4

102.6

Christophersen AS, et al: Determination of a selection of

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C −80°C

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anti-epileptic drugs and two active metabolites in whole

Values are the means of ﬁve replicate determinations.

blood by reversed phase UPLC-MS/MS and some examples of application of the method in forensic toxicology

method described in the current study will be useful for var-cases. Drug Test Anal 7: 634‒644, 2015.

ious studies such as clinical and forensic toxicological11) US FDA: Guidance for Industry: Bioanalytical Method

investigations.

Validation, 2018. https://www.fda.gov/downloads/drugs/
guidances/ucm070107.pdf.

Conﬂict of Interest

12) Dias BCL, Fachi MM, de Campos ML, Degaut FLD,
Peccinini RG, et al: A new HPLC‒MS/MS method for the

The authors declare no conﬂict of interest.

simultaneous quantiﬁcation of SGLT2 inhibitors and met-

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