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INTRODUCTION

It is important to understand the impurity profiles of drug products

and drug substance material. Assessing the purity of the sample

allows pharmaceutical companies to make decisions during the

development and to move forward through commercialization of

the drug. Impurity profiles dictate raw material quality from

vendors, finished product shelf life, route synthesis pathways, and

intellectual protection from counterfeiting.

In this application, the ACQUITY UPC2 System was used to analyze

metoclopramide and related impurities. The method development

investigated columns and solvents to determine suitable method

conditions optimizing specificity and peak shape. During the

method development process, anomalies were observed during the

method development screening process.5 In one instance, a

standard solution of Impurity F was hypothesized to be unstable

after a few days. we use ACQUITY UPC2 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.

ANALYSIS OF METOCLOPRAMIDE AND RELATED IMPURITIES USING UPC

2/MS

Michael D. Jones, Andrew Aubin, Paula Hong, and Warren Potts

Waters Corporation, Milford, MA, USA

METHODS

FINAL METHOD CONDITIONs

System: ACQUITY UPC2 PDA SQD

Column: ACQUITY UPC2 BEH 2-EP 3.0 mm x 100 mm, 1.7 µm

Mobile Phase: A: CO2

B: 1g/L Ammonium formate in 50:50 methanol:acetonitrile

spiked with 3% of formic acid

Wash Solvents: 70:30 Methanol:Isopropanol

Separation Mode: Gradient; 5% to 30%B over 5.0 minutes; 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 minutes

Detection: PDA 3D Channel: PDA, 200-410nm; 20Hz, PDA 2D Channel:

275nm @ 4.8nm Resolution (Compensated 500-600nm, SQD MS:

150-1200Da; ES PosNeg

Make-up flow: N/A

Data Management: Empower® 3 CDS

Method Development

RESULTS

CONCLUSIONS

Achiral analysis of metoclopramide and related substances was

successfully performed using the ACQUITY UPC2 System

Method development was facilitated by understanding the

properties of the impurity structures.

The primary method variables that influenced selectivity,

resolution, and peak integrity were stationary phase, modifier

elution strength, and additive composition, respectively.

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.

investigating instability of Impurity F provided insight to other

potential impurities that may be present in the drug sample

impurity profile.

Interrogation of the UV and MS data was simply performed using

Empower 3 CDS.

Overall, utilizing UPC2/MS increased the knowledgebase about

the pharmaceutical product quality and improved the

methodology procedures involved with achieving the analytical

goals.

The CSH Flouro-Phenyl ligand has properties that can affect the retention

mechanism differently than the other 3 columns used in the screening. The

phenyl ring functionality of the ligand can promote π-π interactions

affecting the retention mechanisms for solutes with conjugated double

bonds. It is possible the CSH Flouro-Phenyl stationary phase has the

capability to discriminate between ortho- and meta- configurations of the

small molecule impurity C structure.

metoclopramide

Impurity Analysis

Figure 1. Column screening results. The modifier (B) was methanol with 2g/L

ammonium formate. 5% to 30% B over 5 min and held at 30% for 1 min.

Effect of additive

Column Screening

Figure 2A. Results when using formic acid only for peaks with hydroxyl (or polyphenols) functionality

such as impurity H tend to benefit from the use of only formic acid,

Figure 2B. Results when combining formic acid and ammonium formate provided the benefits of each additive.

Optimal peak shape for compounds with primary, secondary, and tertiary amine functionality trend from the use of ammonium salt-based additives as with impurity F

Final Methodology

Figure 3. Results of injections of standard mixture and expired metoclopramide

sample performed with the final optimized conditions determined by experiments

varying additive concentrations documented in the “Methods” section.

Figure 4. MS spectral analysis of EP impurity C for the doublet peaks observed

when using the UPC2 CSH Flouro-phenyl stationary phase. Inlay of the

chromatographic trace and structure is provided (upper right).

Investigation of Impurity C

Investigation of Impurity F

The peak shape of impurity F was observed to degrade over time during

the method development process. The working standard was prepared in

methanol. Many of the impurity peaks were products of methylation or

methoxylation. Based on this information, alternative diluents should be

explored to inhibit the likelihood of these transformations.

Name Rt (min) Observed m/z

Δ Mass Proposed transformation

EP Impurity F 2.924 286

Unknown 1 2.268 344 + 58 Da methoxylation + methyl-ation

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

Unknowns 3 & 6 2.680 & 2.886 296 + 10 Da hydrolysis + two methyl-ations

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

Figure 5 and Table 1. MS ES+ TIC of a degraded standard solution of

metoclopramide EP impurity F. Table includes masses found in the degraded

standard solution of metoclopramide EP impurity F.