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METHODS Conditions

System: ACQUITY UPLC H-Class with Column Manager

Column: ACQUITY UPLC BEH C18, 1.7 µm, 2.1 x 50 mm

Mobile phase A: 0.1% (v/v) Formic acid in Water

Mobile phase B: 0.1% (v/v) Formic acid in Acetonitrile

Column Temperature: 30 C

Injection volume: 2 uL

Flow rate: 0.8 mL/min

Isocratic:60% A: 40%B

ACQUITY PDA Detector

Wavelength range: 210-400 nm

Resolution: 3.6 nm

Selected wavelengths: 228 nm, 4.8 nm resolution

Time Constant: Normal

Sampling rate: 20 pts/s

ACQUITY ELSD Detector Isocratic Solvent Manager

Gas: 25 psi Solvent: 0.1% (v/v) formic acid in methanol

Data rate: 10 pps Flow rate: 0.3 mL/min

Nebulizer Mode: Cooling

Nebulizer Temperature: 55 ˚C

Sample Preparation:

Glimepiride and related compounds B and C were purchased from the USP.

All standards were dissolved in 55:45 methanol: water and sonicated.

The drug substance glimepiride was obtained from an outside source. Acid

hydrolysis was conducted at 40 ˚C for 0-7 days . The concentration of acid

was 0.1M HCl in the degradation reaction.

INTRODUCTION

Forced degradation studies are typically performed using

HPLC and UV detectors to understand the degradation

pathway of pharmaceuticals and to insure all impurities

are accounted. In these studies, performing mass

balance or the conservation of mass is crucial. Multiple

orthogonal detectors based on different principles can be

used to measure or identify compounds with different

chemical or physical properties. We will evaluate mass

balance using a triple detection system consisting of a

PDA, ELSD and a mass detector. Relative response ratios

will then be used to perform mass balance. The

degradation path way will then be confirmed using of a

mass detector, specifically through the identification of

impurities and their by-products.

USE OF A TRIPLE DETECTION (UV-ELSD-MS) SYSTEM FOR MASS BALANCE IN FORCED DEGRADATION OF PHARMACEUTICALS Paula Hong and Patricia R. McConville

Waters Corporation, 34 Maple Street, Milford, MA 01757

References

1. Chapter <621> CHROMATOGRAPHY United States Pharmacopeia and National Formulary (USP 37-NF

32 S1) Baltimore, MD: United Book Press, Inc.; 2014. p. 6376-85.

2. Mark AN, Andreas K, Patrick JJ. Role of Mass Balance in pharmaceutical stress testing. Pharmaceutical

Stress Testing: CRC Press; 2011. p. 233-53.

CONCLUSIONS Triple detection system in combination with

Empower 3 FR2 provides various tools to assist in

mass balance, including:

Determination of relative response factors by

using the ratio of UV peak and the log of ELSD

peak responses

The ability to input relative response values into

Empower 3 FR2 to determine corrected area

values for impurities for mass balance

determinations

Figure 1. ACQUITY UPLC H-Class system with triple detection including

ACQUITY PDA, ELSD and QDa detectors. The triple detection system

includes an isocratic solvent manager (ISM) which provides make-up

solvent to the QDa detector and houses the splitter required for the ELSD

and the QDa. After the PDA detector, the flow is split to the ELSD detector

and QDa. The composition and flow rate of the make-up solvent impact the

split ratio to the ELSD and the QDa.

Pre-configured Splitter

in the Isocratic Solvent

ACQUITY UPLC H-Class

System with Column Managers and Triple

Detection (UV-ELSD-PDA)

Flow diagram

Wavelength Separation Conditions

RRF Rel Compound B

RRF Rel Compound C

228 Isocratic 1.36 1.10

Table 1. Relative Response Factors for Related compound B and C using the

ratio of the slope of the API/slope of the impurity. The value for related

compound B is outside of 0.8-1.2 range and, therefore, should be applied, as

specified by the USP Chapter <621>.1

AU

0.00

0.05

0.10

0.15

Minutes

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

LS

U

0.00

10.00

20.00

Minutes

1.00 2.00 3.00 4.00 5.00

Inte

nsity

0.0

5.0x105

1.0x106

1.5x106

2.0x106

Minutes

1.00 2.00 3.00 4.00 5.00

PDA @ 228 nm

ELSD

MS XIC

ESI+

Figure 2. Separation of standards of active pharmaceutical ingredient (API),

related compound B and related compound C under isocratic conditions. The

overlay of standards at 250 µg/mL for the API and 10 µg/mL for related

compounds B and C shows the differences in relative response among the

detectors. The UV and ELSD give similar relative response for the three

compounds. In the mass detector related compound C has a greater peak

area than related compound B.

RESULTS AND DISCUSSION Multi-detection of API and Related Compounds

API Rel Cmpd B

Rel Cmpd C

Figure 4. ELSD calibration curves for glimepiride related compound C. The

ELS detector has a quadratic fit to the calibration curve (left) for peak area

vs. the amount. If the values are converted to the logarithmic functions

(inset), the calibration curve fit is linear (right). The R2 value for this curve

is 0.999140.

Determination of Relative Response Factors

Calibration curve ELSD stack

Project Name: MassBalance Glimep 14Oct15Reported by User: System

Report Method: Calibration curve ELSD stack Date Printed:

24851 2/26/2016Report Method ID: 24851

9:49:19 AM US/Eastern

Calibration Plot

Name: Glimepiride C; Fit Type: Linear (1st Order); R^2: 0.999140; Equation Y = 1.67e+000 X + 2.14e+000

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

7.00

LogAmount

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

Point Information ' Peak: GlimepirideB' table contains no data.

1

2

3

4

5

6

7

8

9

10

Name Level X Value Response Calc. Value % Deviation Manual Ignore

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

1.000000

1.000000

1.000000

1.698970

1.698970

1.698970

2.096910

2.096910

2.096910

2.397940

3.838315

3.805043

3.820003

4.921575

4.942371

4.964010

5.629349

5.648075

5.678314

6.122215

1.018281

0.998353

1.007314

1.667098

1.679553

1.692515

2.091018

2.102234

2.120345

2.386219

1.828

-0.165

0.731

-1.876

-1.143

-0.380

-0.281

0.254

1.118

-0.489

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

Peak: Glimepiride C

Calibration curve ELSD stack

Project Name: MassBalance Glimep 14Oct15Reported by User: System

Report Method: Calibration curve ELSD stack Date Printed:

24851 2/26/2016Report Method ID: 24851

9:50:31 AM US/Eastern

Calibration Plot

Name: Glimepiride C; Fit Type: Quadratic (2nd Order); R^2: 0.996621; Equation Y = 1.62e+001 X̂ 2 + 1.65e+003 X - 2.00e+004

-2.0x105

0.0

2.0x105

4.0x105

6.0x105

8.0x105

1.0x106

1.2x106

1.4x106

1.6x106

Amount

0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00 180.00 200.00 220.00 240.00

Point Information ' Peak: GlimepirideB' table contains no data.

1

2

3

4

5

6

7

8

9

10

Name Level X Value Response Calc. Value % Deviation Manual Ignore

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

Glimepiride C

10.000000

10.000000

10.000000

50.000000

50.000000

50.000000

125.000000

125.000000

125.000000

250.000000

6891.517641

6383.265905

6606.982471

83478.507006

87573.059729

92047.180381

425941.030337

444707.880031

476775.136351

1324998.044518

14.265562

14.025072

14.131038

43.764007

45.086203

46.510456

122.439136

125.740811

131.244015

241.342736

42.656

40.251

41.310

-12.472

-9.828

-6.979

-2.049

0.593

4.995

-3.463

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

No

Peak: Glimepiride CELSD calibration curve

Quadratic fit

ELSD calibration curve

Linear fit

Calibration curve

Project Name: MassBalance Glimep 14Oct15Reported by User: System

Report Method: Calibration curve Date Printed:

23805 12/28/2015Report Method ID: 23805

8:36:43 PM US/Eastern

Calibration Plot

Name: Glimepiride; Fit Type: Linear (1st Order); R^2: 0.996948; Equation Y = 1.84e+000 X + 1.38e+000

Name: GlimepirideB; Fit Type: Linear (1st Order); R^2: 0.999125; Equation Y = 1.69e+000 X + 2.25e+000

Name: GlimepirideB; Fit Type: Linear (1st Order); R^2: 0.999125; Equation Y = 1.69e+000 X + 2.25e+000

Name: Glimepiride; Fit Type: Linear (1st Order); R^2: 0.996948; Equation Y = 1.84e+000 X + 1.38e+000

Name: Glimepiride C; Fit Type: Linear (1st Order); R^2: 0.999140; Equation Y = 1.67e+000 X + 2.14e+000

Name: Glimepiride; Fit Type: Linear (1st Order); R^2: 0.996948; Equation Y = 1.84e+000 X + 1.38e+000

LogA

rea

1.00

2.00

3.00

4.00

5.00

6.00

7.00

ppm

0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

1

2

3

4

5

Name Y-axis X-axis Fit Type R^2 Equation

GlimepirideB

Glimepiride C

Glimepiride

Glimepiride C

Glimepiride

LogArea

LogArea

LogArea

LogAmount

LogAmount

LogAmount

Linear (1st Order)

Linear (1st Order)

Linear (1st Order)

0.999125

0.999140

0.996948

Y = 1.69e+000 X + 2.25e+000

Y = 1.67e+000 X + 2.14e+000

Y = 1.84e+000 X + 1.38e+000

Figure 3. Overlay of glimepiride related compound C standards (10-250 µg/

mL) in PDA and ELSD. The UV detector produces a linear response for

standards. Evaluating the peak areas in the ELSD, a non-linear or logarithmic

response is observed. For example, at 10 µg/mL the response in the ELSD

(pink trace) is significantly lower than that observed in the PDA.

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

Minutes

0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

PDA 250 µg/mL 125 µg/mL 50 µg/mL 10 µg/mL

LS

U

0.00

100.00

200.00

300.00

400.00

500.00

600.00

700.00

800.00

900.00

Minutes

0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

250 µg/mL 125 µg/mL

50 µg/mL

ELSD

Table 3. Mass balance determinations for forced degradation of glimepiride.

The calculations were performed using RRF determined with the ELSD

method. The RRF were entered into Empower 3 FR 2 for corrected values of

the related impurities. All mass balance values were within 2%.

n = 3

Reference

T=0

1 day

3 days

5 days

7 days

Amount

238.4 236.1 239.0 242.7 239.1 244.1

% Recovery

99.0 100.2 101.8 100.3 102.4

AU

0.00

0.20

0.40

374.

3

432.

2

513.

2

AU

0.00

0.20

0.40

AU

0.00

0.10

0.20

0.30

0.40

0.50

Minutes

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50

t=0

t= 3 days

t= 5 days

Figure 5. UV chromatograms of forced degradation of glimepiride drug

substance with base mass labels. The drug substance was exposed to acidic

hydrolysis conditions at 40 C over a period of days. Over the course of the

study the two impurity peaks ( related compound C and B) increased in peak

area.

API

Rel Cmpd B

Rel Cmpd C

Standard

RRF Rel Compound B

RRF Rel Compound C

50 1.25 1.10

125 1.24 1.09

Table 2. Relative Response Factors for related compound B and C using the

ratio of the UV peak area to the log of the ELSD peak area. RRF can be

calculated using the response of the UV detector to a mass concentration

dependent detector.2 This assumes a linear relationship for both detectors.

To convert the ELSD calibration response to a linear function, the log of

both x and y values can be used. Thereby, using the log of the ESLD peak

area, we can calculate RRF factors for both impurities. These values have

good correlation with those obtained using the slopes of the calibration

curves in the UV.

Mass Balance for Forced Degradation Studies

𝑅𝑅𝐹 = [𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝐼𝑚𝑝𝑢𝑟𝑖𝑡𝑦]

[𝑆𝑙𝑜𝑝𝑒 𝑜𝑓 𝐴𝑃𝐼]

𝑅𝑅𝐹 = 𝑈𝑉 𝐴𝑟𝑒𝑎𝐼𝑚𝑝𝑢𝑟𝑖𝑡𝑦

𝐶𝑜𝑛𝑐 𝐷𝑒𝑡𝑒𝑐𝑡𝑜𝑟 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑖𝑚𝑝𝑢𝑟𝑖𝑡𝑦/

𝑈𝑉 𝐴𝑟𝑒𝑎𝐴𝑃𝐼𝐶𝑜𝑛𝑐 𝐷𝑒𝑡𝑒𝑐𝑡𝑜𝑟 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝐴𝑃𝐼

𝑅𝑅𝐹 = 𝑈𝑉𝐴𝑟𝑒𝑎𝐼𝑚𝑝𝑢𝑟𝑖𝑡𝑦

𝐸𝐿𝑆𝐷 log(𝐴𝑟𝑒𝑎 𝑖𝑚𝑝𝑢𝑟𝑖𝑡𝑦 )/

𝑈𝑉 𝐴𝑟𝑒𝑎𝐴𝑃𝐼𝐸𝐿𝑆𝐷 log (𝐴𝑟𝑒𝑎 𝐴𝑃𝐼)

=

10 µg/mL