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52 August 2014Spinco Biotech
HPL
C A
naly
sis
Application Brief
Prominence- embraces EP method-Paracetamol
Paracetamol, also known as Acetaminophen, is a widely used ‘over-the-counter’ analgesic and antipyretic. It is chemically described as N-acetyl -p-aminophenol. It is commonly used for relief of headaches and is the major ingredient in numerous flu and cold remedies. Paracetamol also features in the World Health Organization’s list of essential medicines (under Non opioids and non steroidal anti-inflammatory drugs and anti migraine medicines as of October 2013), a list comprising of the most important medicines required in a basic health system.
Though it is generally safe to consume at recom- mended dosages for a short course of medication, it is more toxic when compared to the other non prescription pain relievers. The side effects of Paracetamol depends on the dosage and course of treatment. Generally under regulated conditions, the effects of Paracetamol are mild and nonexistent. However, in adverse conditions it can lead to liver damage, skin reactions and asthma. Since the drug has a very close safety margin, there is very little difference between recommended therapeutic and potentially toxic dose. Hence there is a need to thoroughly analyze the compounds in accordance with standardized norms.
The Pharmacopoeia (US, European, British etc.) has been described as an important component of quality system that determines the drug safety and efficacy. Its objective is to provide an analytical method to deliver quality drug products. Many of the methods in the existing monograph apply traditional methods or methods that need modernization. Many a time, these methods employ concentrated buffers and longer analysis times, thereby reducing productivity and adding cost. In this report, we present an effective method of analysis for determination of related impurities in Paracetamol using the new Shimadzu Prominence-i HPLC system, that uses mild buffers and shorter analysis time.
HPLC
Shimadzu HPLC Prominence-i LC-2030C innovatively embraces EP method- Paracetamol
Application News
AD-0057Paracetamol, also known as Acetaminophen, is a widely used “over-the-counter” analgesic and antipyretic. It is chemicallydescribed as N-acetyl -p-aminophenol. It is commonly used for relief of headaches and is the major ingredient in numerousflu and cold remedies. Paracetamol also features in the World Health Organization’s list of essential medicines (under Nonopioids and non steroidal anti-inflammatory drugs and anti migraine medicines as of October 2013), a list comprising of themost important medicines required in a basic health system.
Though it is generally safe to consume at recommended dosages for a short course of medication, it is more toxic whencompared to the other non prescription pain relievers. The side effects of Paracetamol depends on the dosage and course oftreatment. Generally under regulated conditions, the effects of Paracetamol are mild and nonexistent. However, in adversey g , ,conditions it can lead to liver damage, skin reactions and asthma. Since the drug has a very close safety margin, there isvery little difference between recommended therapeutic and potentially toxic dose. Hence there is a need to thoroughlyanalyze the compounds in accordance with standardized norms.
The Pharmacopoeia (US, European, British etc.) has been described as an important component of Quality system thatdetermines the drug safety and efficacy. Its objective is to provide an analytical method to deliver quality drug products. Manyof the methods in the existing monograph apply traditional methods or methods that need modernization. Many a time, thesemethods employ concentrated buffers and longer analysis times, thereby reducing productivity and adding cost. In this report,we present an effective method of analysis for determination of related impurities in Paracetamol using the new ShimadzuProminence-i LC-2030C HPLC system that uses mild buffers and shorter analysis timeProminence-i LC-2030C HPLC system, that uses mild buffers and shorter analysis time.
Experimental Results and Discussion
HO
HN
O
CH3
Figure 1: Structure of Paracetamolp
Preparation of Paracetamol and its impurities
Three Paracetamol impurities namely Impurity-K ( p-Aminophenol), Impurity-F(4-Nitrophenol) and Impurity-J(p-Chloroacetanilide) were chosen for this study. Theimpurities mixed standard stock and Paracetamol testsolution were prepared according to EP.5.0monograph.
Method Development
Figure 2 shows the LC Chromatogram of theParacetamol and its impurities run using the methoddeveloped in Prominence-i LC-2030C. The developedmethod employs a gradient elution using mildphosphate buffer that allows shorter runtime for theanalysis of Paracetamol and its impurities. On thecontrary the EP isocratic method uses very long run
Instrument and Analytical Conditions
Table1: Analytical conditions------------------------------------------------------------------------------HPLC : Prominence-i LC-2030CColumn : Shesido Capcell 50x3.0mm,3.0µmWorkstation : Lab solution Version 5.62 PMobile phase-A : 5mM Na2HPO4 + 5mM NaH2PO4 in
waterM bil h B M th l
contrary, the EP isocratic method uses very long runtimes with a broad peak for impurity-J that elutes lastleading to poor sensitivity for this impurity (referFigure-3).
Figure 2: HPLC Chromatogram using the new methodDatafile Name:LC 1107003.lcdSample Name:Paracetamol +Impurity mix
250
300mV
LC 1107003.lcd Detector A 245nm
mol
/3.1
57
Prominence-i HPLC, Developed Method
Mobile phase-B : MethanolGradient : 0.01min(2%), 4.00(15%),10 (50%),
14(50%),16(2%) & 18(2%)Column temp : 35 0C Wavelength : 245nmInjection volume : 5 µL ------------------------------------------------------------------------------
0.0 2.5 5.0 7.5 10.0 12.5 15.0 min
0
50
100
150
200
Impu
rity-
K/0
.994 Par
acet
am
Impu
rity-
F/5.
041
Impu
rity-
J/10
.272
Experimental
Preparation of Paracetamol and its impurities
Three Paracetamol impurities namely Impurity-K (p-Aminophenol), Impurity-F(4-Nitrophenol) and Impurity-J (p-Chloroacetanilide) were chosen for this study. The impurities mixed standard stock and Paracetamol test solution were prepared according to EP.5.0 monograph.
Fig. 1: Structure of Paracetamol
Table1: Analytical conditions
Prominence-i
HPLC
Shimadzu HPLC Prominence-i LC-2030C innovatively embraces EP method- Paracetamol
Application News
AD-0057Paracetamol, also known as Acetaminophen, is a widely used “over-the-counter” analgesic and antipyretic. It is chemicallydescribed as N-acetyl -p-aminophenol. It is commonly used for relief of headaches and is the major ingredient in numerousflu and cold remedies. Paracetamol also features in the World Health Organization’s list of essential medicines (under Nonopioids and non steroidal anti-inflammatory drugs and anti migraine medicines as of October 2013), a list comprising of themost important medicines required in a basic health system.
Though it is generally safe to consume at recommended dosages for a short course of medication, it is more toxic whencompared to the other non prescription pain relievers. The side effects of Paracetamol depends on the dosage and course oftreatment. Generally under regulated conditions, the effects of Paracetamol are mild and nonexistent. However, in adversey g , ,conditions it can lead to liver damage, skin reactions and asthma. Since the drug has a very close safety margin, there isvery little difference between recommended therapeutic and potentially toxic dose. Hence there is a need to thoroughlyanalyze the compounds in accordance with standardized norms.
The Pharmacopoeia (US, European, British etc.) has been described as an important component of Quality system thatdetermines the drug safety and efficacy. Its objective is to provide an analytical method to deliver quality drug products. Manyof the methods in the existing monograph apply traditional methods or methods that need modernization. Many a time, thesemethods employ concentrated buffers and longer analysis times, thereby reducing productivity and adding cost. In this report,we present an effective method of analysis for determination of related impurities in Paracetamol using the new ShimadzuProminence-i LC-2030C HPLC system that uses mild buffers and shorter analysis timeProminence-i LC-2030C HPLC system, that uses mild buffers and shorter analysis time.
Experimental Results and Discussion
HO
HN
O
CH3
Figure 1: Structure of Paracetamolp
Preparation of Paracetamol and its impurities
Three Paracetamol impurities namely Impurity-K ( p-Aminophenol), Impurity-F(4-Nitrophenol) and Impurity-J(p-Chloroacetanilide) were chosen for this study. Theimpurities mixed standard stock and Paracetamol testsolution were prepared according to EP.5.0monograph.
Method Development
Figure 2 shows the LC Chromatogram of theParacetamol and its impurities run using the methoddeveloped in Prominence-i LC-2030C. The developedmethod employs a gradient elution using mildphosphate buffer that allows shorter runtime for theanalysis of Paracetamol and its impurities. On thecontrary the EP isocratic method uses very long run
Instrument and Analytical Conditions
Table1: Analytical conditions------------------------------------------------------------------------------HPLC : Prominence-i LC-2030CColumn : Shesido Capcell 50x3.0mm,3.0µmWorkstation : Lab solution Version 5.62 PMobile phase-A : 5mM Na2HPO4 + 5mM NaH2PO4 in
waterM bil h B M th l
contrary, the EP isocratic method uses very long runtimes with a broad peak for impurity-J that elutes lastleading to poor sensitivity for this impurity (referFigure-3).
Figure 2: HPLC Chromatogram using the new methodDatafile Name:LC 1107003.lcdSample Name:Paracetamol +Impurity mix
250
300mV
LC 1107003.lcd Detector A 245nm
mol
/3.1
57
Prominence-i HPLC, Developed Method
Mobile phase-B : MethanolGradient : 0.01min(2%), 4.00(15%),10 (50%),
14(50%),16(2%) & 18(2%)Column temp : 35 0C Wavelength : 245nmInjection volume : 5 µL ------------------------------------------------------------------------------
0.0 2.5 5.0 7.5 10.0 12.5 15.0 min
0
50
100
150
200
Impu
rity-
K/0
.994
Par
acet
am
Impu
rity-
F/5.
041
Impu
rity-
J/10
.272
HPLC
August 2014 53Spinco Biotech
Results and Discussion
Method Development
Figure 2 shows the LC Chromatogram of the Paracetamol and its impurities run using the method developed in Prominence-i HPLC. The developed method employs a gradient elution using mild phosphate buffer that allows shorter runtime for the analysis of Paracetamol and its impurities. On the contrary, the EP isocratic method uses very long run times with a broad peak for impurity-J that elutes last leading to poor sensitivity for this impurity (refer Figure-3).
Fig 2: HPLC Chromatogram using the new method
Application Brief
HPL
C A
naly
sis
The comparison LC chromatograms for Paracetamol and its related impurities using the EP related substances method as well as the new method developed using the Prominence-i HPLC are as depicted in Figure-4. The comparison of system suitability parameters using the two methods are as presented in Table-2.
Datafile Name:LC 1107012.lcdSample Name:Paracetamol +Impuritymix
Application News
AD-0057
Figure 3: HPLC chromatogram of Paracetamol –EP method Table 2: Chromatographic system suitability criteria forrelated subtances as per EP method
Sample Name:Paracetamol +Impurity mix
0 10 20 30 40 50 min
0
50
100
150
200
250
300
mVLC 1107012.lcd Detector A 245nm
Impu
rity-
K/2
.464
Par
acet
amol
/3.5
78
Impu
rity-
F/1
4.37
0
Impu
rity-
J/42
.565
Impurity-J (Broad Peak)
System suitability EP Method Prominence-iHPLC Method
Resolution between Impurity-K & Paracetamol
Acceptance criteria: minimum 4.0
6.22 91
Signal to Noise Ratio the peak due to
Impurity-J Acceptance criteria:
9.00 149
EP Method
0 10 20 30 40 50 min
The comparison LC chromatograms for Paracetamol andits related impurities using the EP related substancesmethod as well as the new method developed using theProminence-i-LC2030C are as depicted in Figure-4. Thecomparison of system suitability parameters using thetwo methods are as presented in table-2.
Acceptance criteria: Not less than 50
Conclusions
A shorter run time method with greater sensitivity forImpurity-J in Paracetamol has been developedsuccessfully using the new Prominence-i-LC2030Cintegrated LC system The method is much superior to
Figure 4: HPLC comparison Chromatogramintegrated LC system. The method is much superior tothe existing compendial method in terms of increasedefficiency and lower running cost (solvent consumption).
References
[1] European Pharmacopoeia 5.0 01/2005:0049
0
25000
50000
75000
uV
Data2:LC 1107003.lcd Detector A:245nmData1:LC 1107012.lcd Detector A:245nm
EP Method
Prominence-i HPLC Developed Method
10 20 30 40 50 min
0p
SHIMADZU (Asia Pacific) Pte. Ltd79 Science Park Drive, #02-01/08 Cintech IV, Singapore 118264www.shimadzu.com.sgTel: +65-6778 6280 Fax: +65-6778 2050
Copyright © 2013 SHIMADZU (Asia Pacific) Pte. Ltd.All rights reserved. No part of this document may be reproduced in any form or byany means without permission in writing from SHIMADZU (Asia Pacific) Pte. Ltd.
Datafile Name:LC 1107012.lcdSample Name:Paracetamol +Impuritymix
Application News
AD-0057
Figure 3: HPLC chromatogram of Paracetamol –EP method Table 2: Chromatographic system suitability criteria forrelated subtances as per EP method
Sample Name:Paracetamol +Impurity mix
0 10 20 30 40 50 min
0
50
100
150
200
250
300
mVLC 1107012.lcd Detector A 245nm
Impu
rity-
K/2
.464
Par
acet
amol
/3.5
78
Impu
rity-
F/1
4.37
0
Impu
rity-
J/42
.565
Impurity-J (Broad Peak)
System suitability EP Method Prominence-iHPLC Method
Resolution between Impurity-K & Paracetamol
Acceptance criteria: minimum 4.0
6.22 91
Signal to Noise Ratio the peak due to
Impurity-J Acceptance criteria:
9.00 149
EP Method
0 10 20 30 40 50 min
The comparison LC chromatograms for Paracetamol andits related impurities using the EP related substancesmethod as well as the new method developed using theProminence-i-LC2030C are as depicted in Figure-4. Thecomparison of system suitability parameters using thetwo methods are as presented in table-2.
Acceptance criteria: Not less than 50
Conclusions
A shorter run time method with greater sensitivity forImpurity-J in Paracetamol has been developedsuccessfully using the new Prominence-i-LC2030Cintegrated LC system The method is much superior to
Figure 4: HPLC comparison Chromatogramintegrated LC system. The method is much superior tothe existing compendial method in terms of increasedefficiency and lower running cost (solvent consumption).
References
[1] European Pharmacopoeia 5.0 01/2005:0049
0
25000
50000
75000
uV
Data2:LC 1107003.lcd Detector A:245nmData1:LC 1107012.lcd Detector A:245nm
EP Method
Prominence-i HPLC Developed Method
10 20 30 40 50 min
0p
SHIMADZU (Asia Pacific) Pte. Ltd79 Science Park Drive, #02-01/08 Cintech IV, Singapore 118264www.shimadzu.com.sgTel: +65-6778 6280 Fax: +65-6778 2050
Copyright © 2013 SHIMADZU (Asia Pacific) Pte. Ltd.All rights reserved. No part of this document may be reproduced in any form or byany means without permission in writing from SHIMADZU (Asia Pacific) Pte. Ltd.
Datafile Name:LC 1107012.lcdSample Name:Paracetamol +Impuritymix
Application News
AD-0057
Figure 3: HPLC chromatogram of Paracetamol –EP method Table 2: Chromatographic system suitability criteria forrelated subtances as per EP method
Sample Name:Paracetamol +Impurity mix
0 10 20 30 40 50 min
0
50
100
150
200
250
300
mVLC 1107012.lcd Detector A 245nm
Impu
rity-
K/2
.464
Par
acet
amol
/3.5
78
Impu
rity-
F/1
4.37
0
Impu
rity-
J/42
.565
Impurity-J (Broad Peak)
System suitability EP Method Prominence-iHPLC Method
Resolution between Impurity-K & Paracetamol
Acceptance criteria: minimum 4.0
6.22 91
Signal to Noise Ratio the peak due to
Impurity-J Acceptance criteria:
9.00 149
EP Method
0 10 20 30 40 50 min
The comparison LC chromatograms for Paracetamol andits related impurities using the EP related substancesmethod as well as the new method developed using theProminence-i-LC2030C are as depicted in Figure-4. Thecomparison of system suitability parameters using thetwo methods are as presented in table-2.
Acceptance criteria: Not less than 50
Conclusions
A shorter run time method with greater sensitivity forImpurity-J in Paracetamol has been developedsuccessfully using the new Prominence-i-LC2030Cintegrated LC system The method is much superior to
Figure 4: HPLC comparison Chromatogramintegrated LC system. The method is much superior tothe existing compendial method in terms of increasedefficiency and lower running cost (solvent consumption).
References
[1] European Pharmacopoeia 5.0 01/2005:0049
0
25000
50000
75000
uV
Data2:LC 1107003.lcd Detector A:245nmData1:LC 1107012.lcd Detector A:245nm
EP Method
Prominence-i HPLC Developed Method
10 20 30 40 50 min
0p
SHIMADZU (Asia Pacific) Pte. Ltd79 Science Park Drive, #02-01/08 Cintech IV, Singapore 118264www.shimadzu.com.sgTel: +65-6778 6280 Fax: +65-6778 2050
Copyright © 2013 SHIMADZU (Asia Pacific) Pte. Ltd.All rights reserved. No part of this document may be reproduced in any form or byany means without permission in writing from SHIMADZU (Asia Pacific) Pte. Ltd.
Fig. 3: HPLC chromatogram of Paracetamol –EP method
Fig 4: HPLC comparison Chromatogram
Table 2: Chromatographic system suitability criteria for related subtances as per EP method
ConclusionsA shorter run time method with greater sensitivity for Impurity-J in Paracetamol has been developed successfully using the new Prominence-i HPLC integrated LC system. The method is much superior to the existing compendial method in terms of increased efficiency and lower running cost (solvent consumption).
References[1] European Pharmacopoeia 5.0 01/2005:0049
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