FRACTION COLLECTION FOR ISOLATING … COLLECTION FOR ISOLATING IMPURITIES IN FORCED DEGRADATION...
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FRACTION COLLECTION FOR ISOLATING IMPURITIES IN FORCED DEGRADATION STUDIES Paula Hong and Patricia R. McConville Waters Corporation, Milford, MA 01757 TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2017 Waters Corporation INTRODUCTION Forced degradation studies are typically performed to understand the degradation pathway of pharmaceuticals. One of the specific challenges includes determining the response factor of impurities relative to the active pharmaceutical ingredient (API). Incorrectly identifying the relative response factors (RRFs) could lead to over or under quantification of the impurity, which can in turn lead to mass imbalance. In this presentation the impurity will be collected from the forced degradation analysis by small scale fraction collection and subsequently used to determine RRFs. The RRFs will be also evaluated by established methodologies, specifically comparison of the calibration curve of both the API and impurity standard. METHODS Conditions for forced degradation studies: System: ACQUITY UPLC H-Class with PDA and QDa Detector Column: ACQUITY UPLC BEH C18 1.7μm, 2.1 x 50 mm Column Temperature: 30 °C Injection volume: 4 μL (or 10 μL for scale up experiment) Mobile phase (prepared using AutoBlend): B– 125 mM Ammonium hydroxide C– Water; D– Acetonitrile Flow rate: 0.6 mL/min Wavelength: 254 nm MS Settings: Mode: Electrospray (+) Mass range: 50-500 m/z Capillary Voltage: 1.5 kV Cone Voltage:15 V Single Ion Recording channels (SIR): 383.3, 399.3 Make up pump (ISM): 0.3 mL/min, 0.1% formic acid in methanol Conditions for collection scale– up: System: ACQUITY UPLC H-Class with PDA Detector and WFMA Column: XBridge BEH C18 2.5 μm, 3.0 x 75 mm Column Temperature: 30 °C Injection volume: 30.6 μL Mobile phase (same as described for forced degradation) Flow rate: 0.833 mL/min Sample preparation: Oxidation of loratadine drug substance Loratadine and related impurities (n-oxide and epoxide) were purchased from the Toronto Research Chemicals. All standards were dissolved in 1:1 methanol:water and sonicated. The loratadine drug substance was exposed to oxidative conditions (3% H 2 O 2 ) at 70 ˚C for up to 90 minutes. Sample preparation for collected fractions Approximately 70 injections (over a period of 23 hours) of the forced degradation sample were run on a 3.0 x 75 mm column. Both impurity 1 (n- oxide) and impurity 2 (epoxide) were collected and pooled (per peak). The samples were then dried down to remove the organic portion, frozen and then lyophilized. The dried samples were reconstituted with 500 μL of methanol. References 1. Chapter <621> CHROMATOGRAPHY United States Pharmacopeia and National Formulary (USP 37-NF 32 S1) Baltimore, MD: United Book Press, Inc.; 2014. p. 6376-85. CONCLUSION Fraction collection of stressed drug substance can be performed on an analytical scale for multiple peaks in a single analysis Collection and pooling of multiple injections can be used to acquire micrograms of material Collected samples can be used to assess relative response factors by comparison of the standard curves to that of the drug substance. RELATIVE RESPONSE FACTORS To evaluate the relative response factors (RRF) for both related impurities, the standard curves were used. This approach uses the ratio of the slopes of the impurity and API calibration curves. 1 This requires a known amount of sample. For the lyophilized samples, weighing the small amounts of the impurities was challenging. Therefore, the amount of each impurity collected was calculated using calibration curves of the readily available standards. This also allowed for confirmation of the n-oxide and epoxide impurities. RESULTS AND DISCUSSION Time %A %B %C %D 0.00 0 10 60 30 5.00 0 10 35 55 7.00 0 10 10 80 FORCED DEGRADTAION ANALYSIS AND FRACTION COLLECTION Figure 2. Pathway for oxidative degradation of loratadine. Figure 5. Software used to set up fraction collection. Collection Event Table (upper right) allows events to be imported from a processed chromatogram (or result) using Fill From Chromatogram (upper left). In this window a peaks table is populated based on selected result. User has the option to select which peaks to import into collection table. Simulation option allows analyst to visualize peak collection as compared to a chromatogram (lower views). Figure 4 Chromatograms on both a 2.1 x 50mm and 3.0 x 75 mm column, the latter of which was used for fraction collection. Scaling to the 3.0 mm ID column allowed a larger injection volume per injection. Multiple injections were pooled to collect adequate amount of sample. PDA Spectrum PDA 254.0 nm (PDA Spectrum (210-400)nm) 440.2 415.2 399.2 399.2 383.2 AU 0.00 1.00 2.00 3.00 TIC, Smoothed by 5 point Mean Filter., Time offset by -0.010 mins. (QDa Positive(+) Scan (50.00-500.00)Da, Centroid, CV=15) Intensity 0 2x10 7 4x10 7 6x10 7 8x10 7 Minutes 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 Figure 3. Stacked view of UV and mass total ion (TIC) chromatogram of forced degradation of loratadine drug substance with base mass labels. The drug substance was exposed to oxidative conditions at 70 °C for 90 min. A major impurity peak (76% area) was observed at 2.616 min, while a minor peak (0.88% area) was observed at 3.828 min. Both peaks had a base mass of 399.2 suggesting oxidation of the API at a single site. UV @ 254 nm TIC Figure 6. UV chromatogram of lyophilized fractions 1 and 2. Samples were reconstituted in 500 μL of methanol. Quantification of the fractions showed collection of 188 μg of N-oxide and 12 μg of epoxide. Presence of additional peaks in fraction 2 may be attributed to degradation or conversion of the epoxide impurity during lyophilization. Table 1. Calibration curves and relative response factors (RRF) for loratadine and degradation products. Upper limit of calibration curve range for collected samples was limited by the amount of samples. RRF values for both sets of samples were comparable and within acceptable range. Compound Range (μg/mL) R 2 Slope RRF Loratadine 1-500 0.996 16332632 1.0 Standards N-Oxide 1-500 0.998 16150615 1.1 Epoxide 1-500 1.000 3794973 0.2 Collected Fractions N-Oxide 1– 377 0.998 19771229 1.2 Epoxide 0.8-23 1.000 5021606 0.3 Time Total Area (μV*sec) Apparent Mass Balance Total Adjusted Area *(μV*sec) Corrected Mass Balance* Reference 534488 100.0 N/A N/A 30 min 596388 112 582878 109 60 min 604194 113 581550 109 90 min 629781 118 592461 111 Table 2. Comparison of mass balance calculations without and with (*) RRF corrections. Use of RRF resulted in a decrease in mass balance with values closer to 100% than the apparent mass balance. Figure 7. Oxidation of loratadine at various time points over 90 minutes. With increasing time, the drug substance peak area decreased and was accompanied by a significant increase of n–oxide (49% at 90 minutes). The epoxide impurity increased minimally from 0.6 to 0.9 % (see inset). MASS BALANCE To assess the impact of RRF, oxidative degradation of loratadine drug substance was performed at 70 °C. The samples were analyzed after 30, 60 and 90 minutes. Mass balance calculations were performed using the peak area. Peak areas for n-oxide and epoxide impurities were adjusted using the RRF values (*). In general, RRF > 1.0 result in a decrease in peak area, while RRF < 1.0 result in an increase in peak area. Given the relative amount of n-oxide formed in the degradation and its RRF of >1.0, a decrease in mass balance was observed using the RRF values. N- Oxide - 2.616 - QDa Positive Scan Apex 211.9 390.7 399.2 401.2 Epoxide - 3.828 - QDa Positive Scan Apex 266.9 396.3 399.2 401.2 API - 4.854 - QDa Positive Scan Apex 248.5 373.9 383.2 385.2 Figure 1. Waters Fraction Manager - Analytical (WFMA) N-oxide epoxide loratadine loratadine epoxide N-oxide H 2 O 2 H 2 O 2 2.1 x 50 mm, 1.7 μm 10 μL injection Flow rate: 0.6 mL/min 3.0 x 75 mm, 2.5 μm 30.6 μL injection Flow rate: 0.833 mL/min loratadine N-oxide epoxide AU 0.00 0.20 0.40 0.60 0.80 Minutes 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 AU 0.00 0.20 0.40 0.60 0.80 1.00 Minutes 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 N-oxide epoxide loratadine Ref 30 min 60 min 90 min AU -0.002 0.000 0.002 0.004 0.006 Minutes 3.70 3.75 3.80 3.85 3.90 3.95 AU -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 Minutes 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 Fraction 1 n-oxide Fraction 2 epoxide UV @ 254 nm UV @ 254 nm AU 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 AU 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 Minutes 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 1.80 0.018
Transcript of FRACTION COLLECTION FOR ISOLATING … COLLECTION FOR ISOLATING IMPURITIES IN FORCED DEGRADATION...
FRACTION COLLECTION FOR ISOLATING IMPURITIES IN FORCED DEGRADATION STUDIES
Paula Hong and Patricia R. McConville Waters Corporation, Milford, MA 01757
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS
©2017 Waters Corporation
INTRODUCTION
Forced degradation studies are typically performed to understand
the degradation pathway of pharmaceuticals. One of the specific
challenges includes determining the response factor of impurities
relative to the active pharmaceutical ingredient (API). Incorrectly
identifying the relative response factors (RRFs) could lead to over
or under quantification of the impurity, which can in turn lead to
mass imbalance. In this presentation the impurity will be collected
from the forced degradation analysis by small scale fraction
collection and subsequently used to determine RRFs. The RRFs
will be also evaluated by established methodologies, specifically
comparison of the calibration curve of both the API and impurity
standard.
METHODS Conditions for forced degradation studies: System: ACQUITY UPLC H-Class with PDA and QDa Detector Column: ACQUITY UPLC BEH C18 1.7µm, 2.1 x 50 mm Column Temperature: 30 °C Injection volume: 4 µL (or 10 µL for scale up experiment) Mobile phase (prepared using AutoBlend):
B– 125 mM Ammonium hydroxide C– Water; D– Acetonitrile Flow rate: 0.6 mL/min Wavelength: 254 nm
MS Settings: Mode: Electrospray (+) Mass range: 50-500 m/z Capillary Voltage: 1.5 kV Cone Voltage:15 V Single Ion Recording channels (SIR): 383.3, 399.3 Make up pump (ISM): 0.3 mL/min, 0.1% formic acid in methanol
Conditions for collection scale– up:
System: ACQUITY UPLC H-Class with PDA Detector and WFMA Column: XBridge BEH C18 2.5 µm, 3.0 x 75 mm Column Temperature: 30 °C Injection volume: 30.6 µL Mobile phase (same as described for forced degradation) Flow rate: 0.833 mL/min
Sample preparation: Oxidation of loratadine drug substance
Loratadine and related impurities (n-oxide and epoxide) were purchased from the Toronto Research Chemicals. All standards were dissolved in 1:1 methanol:water and sonicated. The loratadine drug substance was exposed to oxidative conditions (3% H2O2) at 70 ˚C for up to 90 minutes.
Sample preparation for collected fractions
Approximately 70 injections (over a period of 23 hours) of the forced degradation sample were run on a 3.0 x 75 mm column. Both impurity 1 (n-oxide) and impurity 2 (epoxide) were collected and pooled (per peak). The samples were then dried down to remove the organic portion, frozen and then lyophilized. The dried samples were reconstituted with 500 µL of methanol.
References 1. Chapter <621> CHROMATOGRAPHY United States Pharmacopeia and National Formulary (USP 37-NF 32 S1)
Baltimore, MD: United Book Press, Inc.; 2014. p. 6376-85.
CONCLUSION
Fraction collection of stressed drug substance can be performed on an analytical scale for multiple peaks in a single
analysis
Collection and pooling of multiple injections can be used to
acquire micrograms of material
Collected samples can be used to assess relative response factors by comparison of the standard curves to that of the
drug substance.
RELATIVE RESPONSE FACTORS
To evaluate the relative response factors (RRF) for both related impurities, the standard curves were used. This approach uses the ratio of the slopes of the impurity and API calibration curves.
1 This
requires a known amount of sample. For the lyophilized samples, weighing the small amounts of the impurities was challenging. Therefore, the amount of each impurity collected was calculated using calibration curves of the readily available standards. This also allowed for confirmation of the n-oxide and epoxide impurities.
RESULTS AND DISCUSSION
Time %A %B %C %D
0.00 0 10 60 30
5.00 0 10 35 55
7.00 0 10 10 80
FORCED DEGRADTAION ANALYSIS AND FRACTION COLLECTION
Figure 2. Pathway for oxidative degradation of loratadine.
Figure 5. Software used to set up fraction collection. Collection Event Table (upper right) allows events to be imported from a processed chromatogram (or result) using Fill From Chromatogram (upper left). In this window a peaks table is populated based on selected result. User has the option to select which peaks to import into collection table. Simulation option allows analyst to visualize peak collection as compared to a chromatogram (lower views).
Figure 4 Chromatograms on both a 2.1 x 50mm and 3.0 x 75 mm column, the latter of which was used for fraction collection. Scaling to the 3.0 mm ID column allowed a larger injection volume per injection. Multiple injections were pooled to collect adequate amount of sample.
PDA Spectrum PDA 254.0 nm (PDA Spectrum (210-400)nm)
440.2
415.2
399.2
399.2
383.2
AU
0.00
1.00
2.00
3.00
QDa Positive Scan MS TIC, Smoothed by 5 point Mean Filter., Time offset by -0.010 mins. (QDa Positive(+) Scan (50.00-500.00)Da, Centroid, CV=15)
Inte
nsity
0
2x107
4x107
6x107
8x107
Minutes
1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50
Figure 3. Stacked view of UV and mass total ion (TIC) chromatogram of forced degradation of loratadine drug substance with base mass labels. The drug substance was exposed to oxidative conditions at 70 °C for 90 min. A major impurity peak (76% area) was observed at 2.616 min, while a minor peak (0.88% area) was observed at 3.828 min. Both peaks had a base mass of 399.2 suggesting oxidation of the API at a single site.
UV @ 254 nm
TIC
Figure 6. UV chromatogram of lyophilized fractions 1 and 2. Samples were reconstituted in 500 µL of methanol. Quantification of the fractions showed collection of 188 µg of N-oxide and 12 µg of epoxide. Presence of additional peaks in fraction 2 may be attributed to degradation or conversion of the epoxide impurity during lyophilization.
Table 1. Calibration curves and relative response factors (RRF) for loratadine and degradation products. Upper limit of calibration curve range for collected samples was limited by the amount of samples. RRF values for both sets of samples were comparable and within acceptable range.
Compound Range (µg/mL)
R2 Slope RRF
Loratadine 1-500 0.996 16332632 1.0
Standards
N-Oxide 1-500 0.998 16150615 1.1
Epoxide 1-500 1.000 3794973 0.2
Collected Fractions
N-Oxide 1– 377 0.998 19771229 1.2
Epoxide 0.8-23 1.000 5021606 0.3
Time Total Area(μV*sec)
Apparent Mass Balance
Total Adjusted Area *(μV*sec)
Corrected Mass Balance*
Reference 534488 100.0 N/A N/A
30 min 596388 112 582878 109
60 min 604194 113 581550 109
90 min 629781 118 592461 111
Table 2. Comparison of mass balance calculations without and with (*) RRF corrections. Use of RRF resulted in a decrease in mass balance with values closer to 100% than the apparent mass balance.
Figure 7. Oxidation of loratadine at various time points over 90 minutes. With increasing time, the drug substance peak area decreased and was accompanied by a significant increase of n–oxide (49% at 90 minutes). The epoxide impurity increased minimally from 0.6 to 0.9 % (see inset).
MASS BALANCE
To assess the impact of RRF, oxidative degradation of loratadine drug substance was performed at 70 °C. The samples were analyzed after 30, 60 and 90 minutes.
Mass balance calculations were performed using the peak area. Peak areas for n-oxide and epoxide impurities were adjusted using the RRF values (*). In general, RRF > 1.0 result in a decrease in peak area, while RRF < 1.0 result in an increase in peak area. Given the relative amount of n-oxide formed in the degradation and its RRF of >1.0, a decrease in mass balance was observed using the RRF values.
N- Oxide - 2.616 - QDa Positive Scan
Apex
211.9
390.7
399.2
401.2
Epoxide - 3.828 - QDa Positive Scan
Apex
266.9396.3
399.2
401.2
API - 4.854 - QDa Positive Scan
Apex
248.5
373.9
383.2
385.2
Figure 1. Waters Fraction Manager - Analytical (WFMA)
N-oxide epoxide loratadine
loratadine
epoxide N-oxide
H2O2 H2O2
2.1 x 50 mm, 1.7 µm 10 µL injection Flow rate: 0.6 mL/min
3.0 x 75 mm, 2.5 µm 30.6 µL injection Flow rate: 0.833 mL/min
loratadine
N-oxide
epoxide
AU
0.00
0.20
0.40
0.60
0.80
Minutes
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00
N-oxide
epoxide
loratadine Ref 30 min 60 min 90 min
AU
-0.002
0.000
0.002
0.004
0.006
Minutes
3.70 3.75 3.80 3.85 3.90 3.95
AU
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
Minutes
2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60
Fraction 1 n-oxide
Fraction 2 epoxide
UV @ 254 nm
UV @ 254 nm
AU
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
AU
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
Minutes
1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80
1.80
0.018
