In this application note, glipizide was subjected to accelerated stress conditions to generate degradation products. The resulting impurities were identified, interrogated and visualised using the UNIFI Scientific Information System.
Forced degradation and impurity identification of glipizide was successfully characterized utilizing UPLC with PDA and HRMS detection on the UNIFI Scientific Information System platform. The ability to acquire accurate MSE data within one injection allowed glipizide and its impurities to be rapidly identified and comprehensively confirmed. UNIFI enabled the use of custom calculations, incorporation of UV (and analogue) traces as well as trend plots to easy visualize and interpret their relationship and stabilities.
UltraPerformance Liquid Chromatography (UPLC), photo diode array (PDA) detection characterisation of both known and unknown impurities is a critical step in the pharmaceutical industry.1, 2 There is a need to more rapidly identify and further characterise these impurities. By employing high resolution mass spectrometry (HRMS) techniques we can establish a comprehensive dual detection and characterization strategy.
A tightly integrated data package for the acquisition, identification, confirmation, interrogation, reporting, and storing information in libraries provides a powerful approach for impurity profiling, and has proven to be exceptionally beneficial to scientists for the characterization of complex samples.
A comprehensive precursor and product ion dataset acquired by use of the MSE acquisition mode is demonstrated. MSE simultaneously acquires full scan precursor and product ion spectra from a single acquisition without the requirement to preselect precursor ions. This simplifies data acquisition because it does not require advanced knowledge of the analytes.3 Additionally, it also ensures all ionizable impurities can be detected, and in most cases identified, in the first acquisition. In this technology brief, glipizide was subjected to accelerated stress conditions to generate degradation products.4 The resulting impurities were identified, interrogated and visualised using the UNIFI Scientific Information System.
Acid and temperature degradation: The acid catalyzed/temperature degradation studies were carried out by adding a small aliquot of formic acid to a solution of glipizide. The sample was then left at 80 °C and aliquots taken at 0, 6, 24, 48, 72, and 96 hour time points. The samples were diluted 1:10 with mobile phase prior to injection.
System: |
ACQUITY UPLC I-Class (FTN) |
Column: |
ACQUITY UPLC BEH C18, 2.1 x 150 mm, 1.7 μm |
Run time: |
11.0 minutes |
Vials: |
Waters Maximum Recovery |
Column temp.: |
45 °C |
Sample temp.: |
8 °C |
Injection volume: |
0.5 μL |
Flow rate: |
0.4 mL/min |
Mobile phase A: |
water + 0.1% formic acid |
Mobile phase B: |
acetonitrile + 0.1% formic acid |
Time |
%A |
%B |
Curve |
---|---|---|---|
0.0 |
95 |
5 |
– |
7.0 |
40 |
60 |
6 |
8.0 |
0 |
100 |
6 |
9.0 |
95 |
5 |
11 |
MS system: |
Xevo G2-XS QTof |
Ionization mode: |
ESI+ |
Source temp.: |
120 °C |
Desolvation temp.: |
450 °C |
Desolvation gas: |
800 L/hr |
Reference mass: |
Leucine enkephalin [M+H]+ m/z 556.27658 |
Acquisition range: |
m/z 50–1200 |
Scan time: |
0.1 sec |
Capillary voltage: |
1.0 kV |
Cone voltage: |
25 V |
Collision energy: |
Function 1: 6 eV |
Function 2: |
Ramped 25–45 eV |
Pathway profiling – UNIFI v1.8.2
In this study, glipizide (shown in Figure 1) was used as a model compound to demonstrate impurity identification using UPLC-MSE and the UNIFI Scientific Information System.5,6 Two main impurities at m/z 379.1074 and m/z 321.1020 were identified and have also been reported in the literature.4 Using accurate m/z values, UNIFI determined and verified the correct molecular formulas C16H18N4O5S and C14H16N4O3S, respectively.
Glipizide and the two impurities identified in the samples by mass, isotopic intensity and isotopic mass are tabulated along with a trend plot (Figure 2). The trend plot details the decrease of glipizide followed by the concomitant riseof the impurities.
As depicted in Figure 3, the spectrum shows the low energy channel containing precursor ion information and the bottom spectrum shows the high energy MSE channel, containing fragment ion information. Product ion assignment is automatically performed during the analysis, annotated directly on the spectrum, greatly simplifying interpretation. In the high energy data, the ion at m/z 321.1019 has been annotated showing the bond cleavage and the charge retaining portion of the molecule.
A custom calculation was used in order to show the % remaining (or conversion of glipizide) over the course of the experiment (Figure 4). The extracted ion chromatogram (XIC) of glipizide in the 24 hr sample (bottom left panel of Figure 4), as well as the PDA trace at 254 nm and a summed trace of all the identified components, is displayed in the component table representing data from all of the injections.
For further characterization, it is possible using the UV or MS, to determine the relative response of the API and any impurities identified within the samples. Custom calculations were implemented (Figures 5 and 6) displaying the percentage of glipizide against the two impurities within the 48 hr sample, respectively.
Forced degradation and impurity identification of glipizide was successfully characterized utilizing UPLC with PDA and HRMS detection on the UNIFI Scientific Information System platform. The ability to acquire accurate MSE data within one injection allowed glipizide and its impurities to be rapidly identified and comprehensively confirmed. UNIFI enabled the use of custom calculations, incorporation of UV (and analogue) traces as well as trend plots to easy visualize and interpret their relationship and stabilities.
720005902, January 2017