using mass spectral detection with usp methods

USING MASS SPECTRAL DETECTION WITH
USP METHODS
Authors: Thomas E. Wheat, Daniel S. Root, Aparna Chavali, Patricia McConville
Affiliations: Waters Corporation, Milford MA, USA
INTRODUCTION
The analytical methods that are included in USP monographs are preferred
for the development and quality control of drug substances and products.
While the chromatographic methods are typically robust and often include
the common related substances, they can be difficult to validate where
confirmation of peak identity is required. Further, identification of
unexpected components can prove difficult. These complications arise
because the specified mobile phases often contain high concentrations of
buffer salts. These conditions compromise ionization for mass spectral
detection and can complicate collection of fractions for use in
complementary techniques. Substantial effort has been expended in many
laboratories in converting these USP methods to mobile phase modifiers
more suitable for MS. This challenging redevelopment of methods has
seldom proven efficient or satisfactory. Further, that process would require
substantial revalidation of the chromatographic method. As a more
practical alternative, a systematic protocol has been developed for using
MS detection directly with these methods and for guiding the transition to
generally useful separations eliminating non-volatile buffers.
METHOD
System
ACQUITY® UPLC System with 2D Technology
Alpha Pump: ACQUITY UPLC H-Class Quaternary Solvent Manager QSM
Beta Pump: ACQUITY UPLC I-Class Binary Solvent Manager BSM
Dilution Pump: ACQUITY Isocratic Solvent Manager ISM
Sample Manager: ACQUITY UPLC I-Class Sample Manager – Flow-ThroughNeedle
Column Manager (CM-A) configured for two Active Pre-Heaters, additional
tubing and 2D valve kit
UV Detection: ACQUITY UPLC Photodiode Array (PDA) Detector 220 nm
Mass Detection: ACQUITY QDa Detector
MassLynx 4.1 SCN 888 with Waters pump control
Figure 1. Plumbing diagram for peak transfer from a USP HPLC
method. Red path indicates flow through the HPLC column.
The blue indicates flow through the UPLC column. MassLynx
system software controls all module and valve events. The ISM
flow combines with the flow from the HPLC column to dilute organic concentration for retention of the transferred peak.
Figure 4. ESI+ SIR confirmation of the identity of Irbesartan
API at m/z 429.2 from peak transfer. The baseline upset at
20.5 min. reflects the flow change with trapping.
Figure 2. Irbesartan USP UV profile. Unidentified peak is most
likely related compound A, but confirmation is needed. This
peak will be transferred to the mass detector.
Figure 5. The ESI+ SIR at m/z 447.1 confirms the transferred,
unidentified peak as related compound A.
Conditions
USP method
HPLC column:
Mobile phase:
Solution:
XSelect HSS T3 4.6mm x 250mm 5 μm (L1)
Buffer Solution and Acetonitrile (60:40), Buffer
0.55% Phosphoric Acid in Water adjusted to pH 3.2
with Triethylamine , Isocratic
Flow rate:
1.0 mL/min
UPLC elution method
UPLC column: BEH C18, 2.1 x 50 mm, 1.7µm
Mobile phases:
Water, Acetonitrile
Dilution flow:
0.1% formic acid in water, 0.2 mL/min
Gradient:
0 to 95% acetonitrile, 3 minutes, 0.5 mL/min
Mass detection
ESI+ SIR Irbesartan m/z 429.2 , and Irbesartan related compound A (RCA)
m/z 447, Full scan m/z 100—500
CONCLUSION
PROTOCOL

USP method is run with UV detection—QSM delivering flow

When peak of interest begins,

flow is diverted to the UPLC Column

flow from the Beta pump is delivered to the HPLC column

flow from the ISM dilutes the flow prior to the UPLC column

flow from the UPLC column is sent to waste

the peak is trapped at the head of the UPLC column

The USP mobile phase is eluted to waste and replaced

With the flow sent to the mass detector, the compound of interest is
eluted from the UPLC column into the QDa mass detector

The QDa detector monitors the eluate using SIR for expected
compounds, with full scan to detect additional species

The fully-automated multi-valve, multi-pump
ACQUITY® UPLC System can be configured to combine
analytical separations

At-column dilution ensures good retention and peak
shape of analytes transferred with this protocol

The described configuration and protocol can be used
for mass spectral detection of analytes separated with
high ionic strength, non-volatile mobile phases
without redeveloping and revalidating the method

This method can be applied to the identification of
minor or unidentified components in pharmaceutical
products
Figure 3. The benefit of at-column dilution (ACD) for the transferred peak of related compound A. Without ACD the peak is
retained but very distorted.
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