1

WAT E R S SO LU T IO NS

ACQUITY UPC2 System

Empower® 3 Software

ACQUITY SQD Mass Spectrometer

K E Y W O R D S

UPC2, pharmaceutical impurities,

stability indicating methods,

degradation profiling, method

development, metoclopramide,

convergence chromatography

A P P L I C AT IO N B E N E F I T S ■■ Solve complex impurity profile challenges

■■ Approaches to aid final

method development decisions

IN T RO DU C T IO N

UltraPerformance Convergence Chromatography™ (UPC2™) exploits the benefits

of sub-2-µm particle size stationary phases, with carbon dioxide as the primary

mobile phase component. Convergence chromatography is a complementary

analytical technique to liquid chromatography as it provides orthogonal

selectivity, thereby increasing the opportunity to identify impurities present

in a sample. Mass spectral information helps analysts confirm, identify, and

characterize the quality of the pharmaceutical ingredients. Coupling UPC2 to mass

spectrometry provides an important tool for pharmaceutical analysis compared

to previously published reversed phase liquid chromatography (RPLC) impurity

analysis approaches.1-3

Anomalies were observed during the method development screening process.4

In one instance, a standard solution of impurity F was hypothesized to be unstable

after a few days. In this application, we use the ACQUITY UPC2™ System coupled

to ACQUITY® SQD to analyze the identity and relationship of the unknown peaks

observed during the method development standards and expired samples of

metoclopramide. Impurity relationship to the API are hypothesized and confirmed

with the use of the MS spectral data. Finally, the MS data from the impurity profile

was interrogated to ensure the specificity of the methodology in the presence of

these unknown peaks to aid future refinement of the final method.

Impurity Profiling Using UPC2/MSMichael D. Jones and Warren PottsWaters Corporation, Milford, MA, USA

CH3

CH3

N

O

NH

CH3ONH2

Cl

Figure 1. Chemical structure of metoclopramide.

2Impurity Profiling Using UPC2/MS

Sample description

Investigations of impurity C and impurity F standards were prepared in methanol

and explored at 0.1 mg/mL concentrations. The expired sample preparation was

extracted using methanol, and prepared at a concentration of 2 mg/mL relative to the

metoclopramide active ingredient.

R E SU LT S A N D D IS C U S S IO N

Prior to analyzing the degraded metoclopramide sample, the MS data was

examined to address questions regarding observations with the chromatography

of impurity reference standards 4-amino-5-chloro-2-methoxybenzoic acid;

“impurity C” and 4-amino-5-chloro-N-2-(diethylaminoethyl0-2-

hydroxybenzamide “impurity F.”

MS investigation of reference standard impurity C

During the screening process, two peaks were separated during the injection of

the impurity C standard using the ACQUITY UPC2 CSH™ Fluoro-Phenyl column.

This phenomenon was not observed with the other stationary phases. MS spectral

analysis of the two peaks showed similar spectra. The MS spectrum indicates

possible dimerization of the analyte. Based on this information, it was determined

that the two peaks were related to each other rather than the second peak being a

contaminant. The rapid determination of this relationship provided direction for

scoping future analysis by MS/MS and accurate mass. The data generated by those

techniques will be more useful in determining the purity of the standard.

E X P E R IM E N TA L

UPC2 conditions

System: ACQUITY UPC2 with

PDA and SQD detection

Column: ACQUITY UPC2

BEH 2-EP

3.0 x 100 mm,

1.7 µm

Mobile phase A: CO2

Mobile phase B: 1 g/L Ammonium

formate in 50:50

methanol/acetonitrile

spiked with

3% formic acid

Wash solvents: 70:30 methanol/

isopropanol

Separation mode: Gradient; 5% to

30% B over 5.0 min;

held at 30% for 1 min

Flow rate: 2.0 mL/min

CCM back pressure: 1500 psi

Column temp.: 50 °C

Sample temp.: 10 °C

Injection volume: 0.5 µL

Run time: 6.0 min

Detection: PDA 3D Channel: PDA,

200 to 410 nm; 20Hz

PDA 2D Channel:

275 nm at 4.8 nm

Resolution

(compensated

500 to 600 nm)

SQD MS:

150 to 1200 Da;

ES PosNeg

Make-up flow: None; (make-up pump

not configured in

flow splitter)

Data management: Empower 3 Software

3Impurity Profiling Using UPC2/MS

Investigation of impurity F working standard solution stability

The peak shape of impurity F was observed to degrade over time, during the method development process.

Unknown peaks would appear in the chromatogram within the course of a week. In addition, the color of the

standard solution changed from a clear solution to a solution with a brownish tint. MS interrogation revealed

the masses listed in Table 1. The masses were correlated to those found to be significant to the UV trace

(not shown) between retention time 2.0 min and 3.5 min. XIC of m/z = 330 and 296 resulted in multiple peaks.

The working standard was prepared in methanol. Many of the impurity peaks were products of methylation or

methoxylation. Based on this information, alternative diluents will be explored to inhibit the likelihood of these

transformations. Presently, the working standard solution shelf life has been decreased to three days until a suitable

diluent study can be performed. The unknown peak #8 in Table 1 with m/z = 258 has the same mass of methyl

4-(acetylamino)-5-chloro-2-methoxybenzoate, commonly referred to as “impurity B.” This unknown peak found in

the impurity F working standard was determined not to be identical to EP impurity B due to differences in retention

time; whereas, impurity B elutes at approximately 0.48 min. The origins of other impurities are still undetermined.

They are hypothesized to be more products of solution instability or present in the reference material.

2.228 Peak 1 - SQ 1: MS Scan 1: 200.00-600.00 ES+, Centroid, CV=Tune - AVG (1.4:2.1;2.4:3.1) Th: 6.000

202.1

204.1

303.3

313.2

327.2

335.3

342.1

344.2349.3

403.2

405.2

407.3

425.2427.2

482.3

Inte

nsity

0.0

7.0x105

1.4x106

2.1x106

2.8x106

2.267 Peak 2 - SQ 1: MS Scan 1: 200.00-600.00 ES+, Centroid, CV=Tune - AVG (1.4:2.1;2.4:3.1) Th: 6.000

202.2 303.3

313.2

327.2

335.2

342.2

344.1

403.2

405.2

406.3407.3

425.2427.2

482.2484.2

Inte

nsity

0.0

7.0x105

1.4x106

2.1x106

2.8x106

m/z200.00 240.00 280.00 320.00 360.00 400.00 440.00 480.00 520.00 560.00 600.0

Peak 1

Peak 2

2.2

28

2.2

70

Inte

nsity

0.0

6.0x106

1.2x107

1.8x107

2.4x107

Minutes

2.016 2.064 2.112 2.160 2.208 2.256 2.304 2.352 2.400 2.448

O H

O

C H 3ONH 2

Cl

Figure 2. MS spectral analysis of EP impurity C for the doublet peaks observed when using the UPC2 CSH Fluoro-Phenyl stationary phase.

Name Rt (min) Observed m/z Δ Mass Proposed transformation

EP Impurity F 2.924 286

Unknown 1 2.268 344 + 58 Da methoxylation + methylation

Unknowns 2 & 4 2.303 & 2.614 330 + 44 Da methoxylation

Unknowns 3 & 6 2.680 & 2.886 296 + 10 Da hydrolysis + two methylations

Unknown 5 2.864 356 + 70 Da ?

Unknown 7 3.113 252 - 34 Da Loss of Cl-

Unknown 8 3.288 258 - 28 Da Loss of two CH3 groups

Table 1. Masses found in the degraded standard solution of metoclopramide EP impurity F.

4Impurity Profiling Using UPC2/MS

Investigating an expired metoclopramide sample

The ACQUITY UPC2 System coupled to the ACQUITY SQD Mass Spectrometer controlled by Empower 3

Software provided a simple solution to profile impurities in an expired metoclopramide sample. MS spectral

extraction of peaks in the UV chromatographic trace were simply performed by using right mouse click in

the review window to rapidly confirm known impurities and identify 12 unknown impurities, as shown in

Table 2. The sensitivity of UPC2 provided s/n ≥ 10 for impurities detected with area% ≥ 0.05% in the UV

chromatographic trace. The expired metoclopramide sample was interrogated to determine if the masses

in Table 1 were present. The MS data confirmed the presence of 4 out of the 8 impurities related to EP F;

m/z =296, 344, 252, and 258. In addition to the known impurities, a total of 7 unknown impurities were

detected. The masses, retention time, and UV signal-to-noise were recorded in Table 2.

MS TIC - SQ 1: MS Scan - 1: 200.00-600.00 ES+, Centroid, CV=Tune

Inte

nsi

ty

0.0

4.0x107

8.0x107

1.2x108

1.6x108

Minutes0.00 0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 5.40 6.00

m/z

=28

6

m/z

=34

4m

/z=

330

m/z

=33

0

m/z

=35

6

m/z

=29

6

m/z

=25

2

m/z

=25

8

m/z

=29

6

CH3

CH3

N

O

NH

OHNH2

Cl

Impurity F: [M+H] = 286

Figure 3. MS ES+ TIC of a degraded standard solution of metoclopramide EP impurity F.

5Impurity Profiling Using UPC2/MS

: Aged Metoclop : 50:50 MeOH ACN 1g/L ammonium formate 3% formic acid

0.000

0.035

0.070

0.105

0.140

Minutes0.00 0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 5.40 6.00

Impu

rity

B

Unk

now

n m

/z=

22

6

Impu

rity

9

Impu

rity

D

Me

tocl

op

ram

ide

Impu

rity

G

Impu

rity

H

Unk

now

n m

/z=

26

6

Impu

rity

A

Impu

rity

F

*No

MS

Unk

now

n m

/z=

34

4

Unk

now

n m

/z=

31

0

Unk

now

n m

/z=

27

2

AU

Figure 4. UV 275 nm chromatographic trace of a degraded sample of metoclopramide. The major impurities are labeled.

Rt % Area USP s/n m/z

Impurity B 0.49 0.48 148 258

Unknown #1 0.90 0.34 54 226

Impurity “9” 1.18 1.34 255 (ESI-) 180

Impurity D 1.41 1.14 229 224

Unknown #2 2.18 0.12 22 306

Unknown #3 2.33 0.02 8 358

Unknown #4 2.38 1.29 195 n/a

Impurity A (Isomer 1) 2.49 0.28 48 342

Impurity A (Isomer 2) 2.53 0.41 65 342

Rel. Impurity F (Unk. #1) 2.61 0.69 87 344

Rel. Impurity F (Unk. #6) 2.83 0.06 10 296

Impurity F 2.91 0.51 82 286

Rel. Impurity F (Unk. #7) 3.09 0.14 20 252

Unknown #5 3.14 0.55 77 310

Rel. Impurity F (Unk. #8) 3.28 0.14 20 258

Impurity G 3.37 0.87 130 316

Metoclopramide 3.43 86.07 8186 300

Unknown #6 3.60 2.83 380 266

Unknown #7 3.63 1.09 165 272

Impurity H 4.53 1.62 101 (ESI-) 194

Table 2. Peak list of the UV 275 nm chromatographic trace relative to the injection of the metoclopramide expired sample.

Waters Corporation34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, Empower, and ACQUITY are registered trademarks of Waters Corporation. UltraPerformance Convergence Chromatography, UPC2, CSH, and T he Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2013 Waters Corporation. Produced in the U.S.A.February 2013 720004575EN AG-PDF

CO N C LU S IO NS

The ACQUITY UPC2 System coupled to MS provided a

comprehensive approach to impurity profiling. This configuration

enables a scientist to quickly answer questions regarding reference

standard purity, as shown with reference standard impurity C.

UPC2/MS guided the decisions to investigate diluent choices for

the impurity F working standard and adjusting the shelf life of the

working standard solution. In addition, investigating instability of

impurity F provided insight into other potential impurities that may

be present in the drug sample impurity profile. Furthermore, seven

unknown impurities were detected in the expired metoclopramide

sample. Interrogation of the UV and MS data was simply performed

using Empower 3 Software. Overall, utilizing UPC2/MS increased

the knowledge base of pharmaceutical product quality, and

improved the methodology procedures involved with achieving

the analytical goals.

References

1. Ahuja S, Alsante K. Handbook of Isolation and Characterization of Impurities in Pharmaceutical Compounds. Elsevier. 2003.

2. Jones MD et al. Identification and Characterization of an Isolated Impurity Fraction: Analysis of an Unknown Degradant Found in Quetiapine Fumarate. Waters Application Note 720003079EN. 2009 Oct.

3. Jones MD et al. A Workflow Approach for the Identification and Structural Elucidation of Impurities of Quetiapine Hemifumarate Drug Substance. Waters Application Note 720003081EN. 2009 Oct.

4. Jones MD, Potts W. UPC2 Method Development for Achiral Impurity Analysis. Waters Application Note 720004577. 2013 Jan.