Post on 03-Nov-2020
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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.