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table 2. UPLC Method Validation
ABSTRACT
METHODS
Validation of the Catalent UPLC Method
Purpose
table 1. Chromatographic Conditions of In-house HPLC Method / BP HPLC Method / Catalent UPLC Method
UPLC method was developed and fully validated. No interference
Adrenaline injection is mainly used to treat allergic reactions (anaphylaxis) and cardiac resuscitation. The currently used HPLC method for the determination of related compounds in adrenaline injection (resulting both from adrenaline degradation and from manufacturing process) was not efficient in regard to the resolution of one critical impurity, the adrenaline sulfonate (Fig. 1). The British Pharmacopoeia HPLC method is validated only for the determination of noradrenaline. This project was aimed to develop and fully validate an UPLC method (Fig. 3) which was believed superior in separation and performance than the BP HPLC method (Fig. 2).
Methods
In-house HPLC
Name
Adrenaline
Molecular formula
C9H13NO3
Molecular mass
IUPAC structure
Adrenaline sulfonate
C9H13NO5S
C4H12HSO4 25mM
NaH2PO4-H2O 10mM
Ratio MeOH:Buffer
15:85
5:95
5:95
pH
3.8
3.5
3.8
Mode
Isocratic
Isocratic
Gradient
Column
Alltima C8 5.0 µm, 4.6 x 150mm
Nucléosil C18 5.0 µm, 4.6 x 100mm
Acquity UPLC HSS T3 1.8 µm, 2.1 x 100mm
Flow Rate (mL/min)
2
2
0.5
Mobile phase
Injection Volume (µL)
20
20
2
Run Time (min)
15
15
6.5
Column Oven Temperature
Ambient
Ambient
50°C
Sample Temperature
Ambient
Ambient
2-8°C
A3
A4
Noradrenaline
Adrenalone
Unknown
C8H11NO3
The validated UPLC method provides better selectivity separation
and accuracy than the HPLC methods. The resolution of adrenaline sulfonate increases from less than 1.6 to greater than 2.5 (Table 3) while the total run time is reduced by 57%, from 15 minutes for HPLC to 6.5 minutes for UPLC.
RESULTS
183.2
figure 2. Chromatogram from BP HPLC method for A2 assay
figure 3. Chromatogram from Catalent UPLC method for A1, A2, A3 and A4 assay
247.3
181.2
C9H10NO6S
260.0
A2
A3
A4
Accuracy (Recovery)
97-99%
90-93%
95-99%
ND
Repeatability (RSD)
0.99
>0.99
>0.99
Range (μg/mL)
4-16
1-4
0.5-2
ND
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Methods
In-house HPLC
BP HPLC
Catalent UPLC
USP Resolution Preservative-A1
0.4
1.6
2.6
Run Time (min)
15
15
6.5
Detection of Related Compounds of Adrenaline
3
3
4
CONCLUSIONS A UPLC method was developed and validated. The method was shown to provide better separation than HPLC methods, while reducing the run time from 15 minutes to 6.5 minutes. The method may be used to quantify related compounds and degradents in adrenaline injection for final release and stability studies.
169.2
C9H11NO3
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A1
table 3. Methods Performances Comparison
Methods Comparison
205
Zoom
A2
Catalent UPLC
NaH2PO4-H2O 50mM
figure 1. Chromatogram from In-House HPLC method for A1, A2 and A3 assay A1
BP HPLC
Buffer
Wavelengths (nm)
Definitions Abbreviations
Chromatographic conditions
was observed between the mobile phase, the excipients and the related compounds, indicating the specificity of the method. Acceptable accuracy (Recovery: 90-99%), repeatability (Relative Standard Deviation
