52 August 2014Spinco Biotech

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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

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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