CHAPTER-5shodhganga.inflibnet.ac.in/bitstream/10603/4552/14/14_chapter 5.pdf · Cefamezin,...

144

CHAPTER-5

Evaluation and Method Validation of Low

Molecular weight Organic acids in

Pharmaceutical Drug Substances by

Capillary Electrophoresis

145

5.1 Introduction

A number of organic acids are present as a counter ions or

residual amount in drug substances, which are used during the

manufacture of the drug substances. Generally the organic acids are

used for pH-controlled reaction or introduced into the side chain in

the drug molecules. It may be also a by-product during the drug

synthesis, which makes significant presence as residual impurity in

the drug substances. These residual organic acids are tent to react

with the basic drug molecules and form undesirable products which

will hamper the quality of the drug product or which may act as

catalyst to facilitate the decomposition of the drug substances during

the storage.

Simple low molecular weight organic acids such as citric,

maleic, tartaric, succinic and acetic acids have been quantified by

capillary electrophoresis in a variety of sample types including fruit

juices and wines1-5. These organic acids have limited UV absorption

and are often detected in CE using indirect UV detection. However,

use of short UV wavelengths such as 190 nm can permit to direct

detection of selected simple organic acids6,7. Simple organic acids are

frequently employed as counter-ions for basic drugs, the more popular

acidic salts being maleates, succinates and acetates8.

The importantance of analytical method for quantifying the

organic acids in the pharmaceutical drug substances should be a

146

simple, fast and robust. Generally ion chromatography (IC) has been

used for determining the organic acids in various matrices. In recent

years, it was expected that CE might offer a simple and inexpensive

alternative to IC for determination of organic acids in variety of the

samples. Capillary zone electrophoresis (CZE) is gradually gaining

acceptance as an alternative and complementary technique to high-

performance liquid chromatography (HPLC) for the pharmaceutical

analysis. The principle advantages of CZE include, among others, high

separation efficiency, improved selectivity, low operational cost and

speed of analysis9. CE method has been used to quantitatively

determine both cationic and anionic ions in the drug substances.

Metal ions of acidic drugs such as Na +, K + or Ca 2+ have been

quantified10-12 and inorganic anion ions12 of basic drugs such as C1-,

SO42- have also been determined. In this aspect, it is good to assess

the use of CE with indirect UV detection as a suitable method for

stable, reproducable method for the content of residual organic acid in

pharmaceutical drug substances.

Nowadays, capillary electrophoresis (CE) has been recognized as

a promising technique for the separation and quantification of organic

acids in various matrices13-21. Complementary to established

chromatographic methods, like ion-exclusion chromatography22-27

(IEC), CE provides advantageous features, including short analysis

time, low buffer and sample consumption and low detection limits28.

In my study, a CE method is applied for the determination of some

147

short-chain acids such as formic acid, acetic acid, trifluroacetic acid,

succinic acid, methanesulfonic acid and pivalic acid in

pharmaceutical drug substances such as atorvastatin calcium,

cefazolin, lamivudine and lopinavir. These organic acids are used in

the process of manufacturing these drug substances which may not

be completely removed during the process. It is necessary for

evaluating these residual organic acids with a suitable analytical

method. For evaluating this type of UV inactive substances various in-

direct UV detection methods have been employed using various

background electrolytes such as chromate29, 3,5-dinitrobenzoic acid30,

salicylic acid31 and 2,6- pyridine dicarboxylic acid32 (PDC).

The reaction of short chain organic acid with basic drug

substances or a basic group present in the drug substances is also

quite possible during the storage as well as synthesis which lead to an

undesirable byproduct. Therefore, short chain organic acids are

observed to be a potential impurity of drug substances, and to be

monitored before its release to market. The ICH guideline on

impurities describes that any impurity other than active moiety are to

be controlled with suitable limits in the drug substance irrespective of

its harmful nature33.

Atorvastatin34 is marketed by Pfizer with the trade name Lipitor.

Atorvastatin belongs to the drug class known as statins. Atorvastatin

is helpful to lowering blood cholesterol and stabilizes plaque and

148

prevents strokes through anti-inflammatory and other mechanisms. It

also inhibits HMG-CoA reductase, an enzyme which is present in liver

tissue for the production of cholesterol in the body.

The preparation of atorvastatin calcium involves the

condensation of diketoamide and amino butyl dioxanate in presence of

pivalic acid followed by hydrolysis of the protected group of the

atrovastatin. The atrovastatin acid is treated with sodium hydroxide to

form atrovastatin sodium followed by treatment with calcium acetate

to form atrovastatin calcium abd by-product is sodium acetate. The

Low molecular weight organic acid such as pivalic acid and acetic

acid were used in the preparation of atorvastatin calcium must be

determined its presence or absence in the drug substances. The

synthesis of the atrovastatin is illustrated in Fig 5.1.

149

Fig: 5.1 Synthetic scheme for preparation of Atrovastatin Ca.

NH

O O

O

CH3CH3

F

+

1. Pivalic acid

2.HCl

3.NaOH

4. Ca(CH3COO)

2

NH

O

CH3CH3

F

N

O-

OOH OH

O

CH3

CH3

CH3

OO O

CH3CH3

NH2

2

Ca2+

Diketoamide

Aminobutyldioxonate

Atorvastatin Calcium

CH3COONa+

Lopinavir35 is marketed in combination of other drug

substances and it may available with the trade name of Kaletra and

Aluvia. Lopinavir belongs to an antiretroviral of the protease inhibitor

class.

150

Lopinavir is prepared by debenzylation of diprotected Lopinavir

intermediate with Pd/C in presence of ammonium formate and

generate to the active amine.

Fig: 5.2 Synthetic scheme for preparation of Lopinovir

HCO2NH4

Pd/H2

CH3

O

O

OH

CH3

+

NHN

OH3C CH3

OOH

+

Lopinavir

Diprotected Lopinavir Intermediate

2,6 dimethyl phenoxyacetic acid

BOC protected Lopinavir aminoalchol

Pyrimidine acetic acid derivative

Trifluoroacetic acid

N

NHOH

BOC

NH2

NH

BOC

OH

NH

NH

BOC

OHOCH3

O

CH3

CH3

O

CH3

NH

NH2OHO

NH

NHOHO

NHN

OH3C CH3

O

H3C

CH3

O

Lopinavir Aminoalchol

Active amine

151

The active site of the amine group is then react with 2,6

dimethylphenoxy acetic acid to form BOC protectec lopinavir

aminoalchlol. The protected group of BOC is elimated in presence of

trifluoroacetic acid and gives a lopinavir aminoalchol. This lopinavir

aminoalchol is then treated with tetrahydro--(1-methylethyl)-2-oxo-

1(2H)-pyrimidineacetic acid to form a lopinavir. The presence or

absence of these organic acid, formic acid and trifluroacetic acid

should be evaluated with a suitable analytical method. The synthesis

Scheme of the Lopinavir is shown in Fig 5.2.

Lamivudine36 is a 2',3'-dideoxy-3'-thiacytidine and commonly

called as 3TC. It sold by GSK as Heptovir, Zeffix, Epivir, and Epivir-

HBV. Lamivudine is used for treatment of chronic hepatitis B at a

lower dose than for treatment of HIV.

Lamivudine is synthesis by reduction of the lamivudine ester in

presence of sodiumborohydride and the resulting reaction mixture is

treated with succinic acid to form a lamivudine succinnate. The

succinate salt is then treated with triethylamine to remove the

succinic acid and recrystallized with solvent and obtains a pure

lamivudine. The succinic acid was used in the penultimate step of

lamivudine prepartion, so this impurity must quantify for the quality

of the drug substance. The reaction scheme for the preparation of

lamivudine is demonstrated in figure 5.3

152

Fig: 5.3 Synthetic scheme for preparation of Lamivudine.

O

CH3 CH3

CH3

N

N

O

O

S

NH2

O

NaBH4

Succinic acid

N

N

O

O

S

NH2

OH

OHO

OHO

N

N

O

O

SNH2

OH

Lamivudine

.

Coupled ester

Lamivudine Succinate

Cefazolin37 also called as cephazolin or cefazoline, which is a

first-generation cephalosporin antibiotic and is administrated by

either intramuscular injection or intravenous infusion. Cefazolin is

mainly used to treat bacterial infections of the skin. It can also be

used to treat moderately severe bacterial infections involving the lung,

bone, joint, stomach, heart valve, and urinary tract. Cefazolin is sold

under the different brand names: Novaporin, Cefacidal, Ancef,

Cefamezin, Kefzolan, Cefrina, Elzogram, Faxilen, Gramaxin, Cefacidal,

Kefazol, Kefol, Kefzol, Zinol Kezolin, Reflin, and Zolicef.

Acetic acid is used in the preparation of TAC and TZA. The

pivolyl chloride is used in the preparation of the mixed anhydride and

pivalic acid is a by-product. The TAC and Mixed anhydride

153

compunds are react to form cefazolin acid. The acetic acid and

pivalic acid need to be quantify. The simple reaction scheme is

represented in the Fig. 5.4

Fig: 5.4 Reaction Scheme of Cefazolin acid

N

N

N

N

OOH

NNN

N

O

O

CH3

CH3

CH3

O

Pivolyl chloride

+

N

S

N

CH3

S N

S

OHO

NH

O

N

N

N

N

O

H

N

S

N

CH3

S N

S

OHO

NH2

O

H

Cefazolin acid

1H-Terazoyl-1-yl Acetic acid

(TZA)

Mixed anhydride 7-Amino-3-[[5-methyl1,3,4thiadiazol-2yl

thio]methyl]-3-cephem-4-carboxylic acid

(TAC)

The methanesulfonic acid is used in one of the intermediates

of atorvastatin calcium, lamivudine and lopinavir. Eventhough,

methanesulfonic acid is used in the very early stages, it may carry

forward in trace amounts in the drug substances. So it required an

effective method to quantify this impurity also.

Since Low molecular weight carboxylic acids interact with drug

products by three mechanisms.

154

(1) They induce a change in acid content of formulation, which

can shift the formulation into a less stable pH region and initiate or

accelerate the degradation of the API (Active Pharmaceutical

Ingredient).

(2) Carboxylic acid can react with an amine or alcohol functional

groups of the drug molecule or excipients to give amides and esters

respectively.

(3) Salt formation between low molecular weight carboxylic acid

and basic drugs can influence the solubility, dissolution or chemical

stability of the drugs.

Hence, it is very important to quantify these organic acids such

as formic acid, acetic acid, methanesulfonic acid, trifluoroacetic acid,

succinic acid and pivalic acid in pharmaceutical drug substances

wherever these organic acids are used in the preparation.

In this work, potassium phthalate as background electrolyte

and cetyltrimethylammonium bromide (CTAB) as electro osmotic flow

(EOF) modifier was chosen. The determination of short chain organic

acid such as formic acid, acetic acid, methanesulfonic acid, succinic

acid, and trifluroacetic acid in various drug substances were employed

in indirect UV detection mode in capillary electrophoresis method.

155

This method was validated as per the ICH guideline to prove its

suitability for routine analysis.

5.2 Materials and Instrumentation

5.2.1 Chemicals and Reagents

Formic acid, acetic acid, propionic acid, trifluroacetic acid,

methane sulfonic acid, succinic acid potassium hydrogen phthalate

methanol, sodium hydroxide and cetyltrimethylammonium bromide

(CTAB) were procured from E.Merck, (Mumbai, India) and water was

purified with milli–Q purification system (Millipore, Billerica, MA).

Pivalic acid was purchased from Fluka (German, Melbourne).

Atorvastatin and its related substances, Cefazolin sodium and its

related substances, lopinavir and its related substances and

lamivudine and its related substances were prepared in aurobindo

pharma ltd., India.

5.2.2 Solution preparation

5.2.2.1 Internal Standard solution (IS solution) preparation:

Dissolve 50mg of propionic acid in 2000mL of acetonitrile and

water in the ratio of 1:1v/v.

5.2.2.2 Standard solution preparation

Standard solution was prepared by dissolving accurately

weighed 250mg each organic acids (Acetic acid, formic acid,

trifluroacetic acid, methanesulfonic acid, pivalic acid and succinic

156

acid) in 100mL of IS solution Further, 10mL of this solution was

diluted to 100mL with IS solution. Finally, dilute 10mL of this solution

to 100mL with IS solution and filtered through 0.22m or finer

porosity membrane filter.

5.2.2.3 Sample solution preparation

Sample solution was prepared by dissolving accurately weighed

250mg of drug substance in 10mL of IS solution, and filtered through

0.22m or finer porosity membrane filter. Atorvastatin calcium has a

limited solubility in diluent and for a complete dissolution of the

sample, the concentration was reduced to 10mg/mL.

5.2.2.4 Background electrolyte preparation

Background electrolyte consists of 5mM potassium hydrogen

phthalate and 1.2mM of CTAB ((255mg of potassium hydrogen

phthalate dissolved and 110mg of CTAB in 250ml of water) adjusted

to pH 6.5 with 0.1M sodium hydroxide solution.

5.2.3 Procedure

An Agilent instrument CE system equipped with a diode array

detector along with chemstation software for data acquisition and

processing was used. Separation was carried out in fused silica

capillary with extended light path length (Agilent, Germany) with

effective length of 56cm and i.d. of 50m.

157

The sample and standard solutions were introduced by

hydrodynamic pressure of 50 mbar for 5 sec. The separation was

carried out with constant applied voltage of (-) 25kV at ambient

temperature (25°C). Before introducing sample into the capillary, the

capillary was conditioned with background electrolyte for 3min at the

inlet pressure of 5bar for 3min.

The analytes were detected by indirect UV photometric method

and the wavelength was set at 330nm against reference signal at

210nm. Further the performance of the method was further improved

by the use of internal standard to control both injection precision and

linearity.

5.2.4 Capillary Conditioning:

Before starting the experiment every day, the capillary was

flushed with water for 5min and 0.1M sodium hydroxide solution for

5min. Then rinse the capillary with water for 5min followed by

background electrolyte for 15min at a pressure of 5bar.

New capillaries were rinsed with 0.1M sodium hydroxide for

15min and water for 5min followed by conditioning with background

electrolyte for 15min at a pressure of 5bar.

5.2.5 Evaluation of System Suitability:

158

Inject the standard solution in replicate. RSD for replicate

injections of standard solution is not more than 5.0%.

Electrophoretic Conditions:

Capillary : G 1600-61232 (by Agilent Technology)

Capillary temperature : 25°C

Applied voltage : 25kv

Injection : 50 mbar for 5 sec

Wavelength : Signal 330 nm and Reference 210 nm

Precondition time : 3min ( with Hi-flush, at 5bar pressure)

Stop time : 8min.

Detection : Indirect UV photometric mode

5.2.6 Calculation:

AT1 DS1 Formic acid = ------ × ---------- × P1

(% w/w,) AS1 DT1

AT2 DS2

Acetic acid = ------ × ---------- × P2

(% w/w,) AS2 DT2

AT3 DS3

Methane sulfonic acid = ------ × ---------- × P3

(% w/w,) AS3 DT3

AT4 DS4

Trifluroacetic acid = ------ × ---------- × P4

(% w/w,) AS4 DT4

AT5 DS5

Succinic acid = ------ × ---------- × P5

159

(% w/w,) AS5 DT5

AT6 DS6 Pivalic acid = ------ × ---------- × P6 (% w/w,) AS6 DT6

Where

AT1 = Area ratio of peak due to formic acid and peak due to propionic acid in

sample solution

AS1 = Area ratio of peak due to formic acid and peak due to propionic acid in

standard solution

DS = Dilution factor for the standard solution (weight / dilution)

DT = Dilution factor for the sample solution (weight / dilution)

P1 = Percent potency of formic acid used.

AT2 = Area ratio of peak due to acetic acid and peak due to propionic acid in

sample solution

AS1 = Area ratio of peak due to acetic acid and peak due to propionic acid in

standard solution

P2 = Percent potency of acetic acid used

AT3 = Area ratio of peak due to methane sulfonic acid and peak due to propionic

acid in sample solution

AS3 = Area ratio of peak due to methane sulfonic acid and peak due to propionic

acid in standard solution

P3 = Percent potency of methane sulfonic acid used

AT4 = Area ratio of peak due to trifluroacetic acid and peak due to propionic

acid in sample solution

AS4 = Area ratio of peak due to trifluroacetic acid and peak due to propionic acid

in standard solution

P4 = Percent potency of trifluroacetic acid used

AT5 = Area ratio of peak due to succinic acid and peak due to propionic acid in

sample solution

AS5 = Area ratio of peak due to succinic acid and peak due to propionic acid in

standard solution

P5 = Percent potency of succinic acid used

AT6 = Area ratio of peak due to pivalic acid and peak due to propionic acid in

sample solution

AS6 = Area ratio of peak due to pivalic acid and peak due to propionic acid in

standard solution

P6 = Percent potency of pivalic acid used

160

5.3 Method development

Short chain organic acids are ionized to form an anionic species

in dilued aqueous solution, and are inert to UV photometric

absorption.

The initial method was employed with chromate buffer, for

better and faster analysis a long-chain cationic surfactant CTAB was

added to the electrolyte to form a positively charged surface coating.

The positively charged surface generates an electro osmotic flow (EOF)

in the same direction as the migration of organic acids, which

significantly improves the peak shape and reduces the analysis time.

A constant level of EOF is essential on the capillary wall for

reproducible results. A 0.25mM of CTAB was introduced in 5mM of

chromate buffer and pH was adjusted to 8.0 with sodium hydroxide.

The temperature of the capillary was maintained at 25°C and applied

voltage was (–) 25kV. A low conductivity buffer of boric acid was also

introduced in the background electrolyte for stable background. The

peak shapes of organic acids were not in good and no desirable

separations between the organic acids were achieved in this trail.

161





Polarity : Negative



Precondition time : 3min (with Hi-flush, at 5bar pressure)

Stop time : 10min.


Buffer :5mM of Sodium chromate

and 0.5mM of CTAB

pH : 8.0 with NaOH

In another trail a 5mM of Benzoic acid buffer contains 5% of

methanol with pH 6.0 was used. The EOF was reversed by using the

0.5mM of CTAB. This trail also did not improve the peak shape and

resolution of the organic acids.

162

Next trail was utilized with 5mM of phthalate additive and

0.5mM of CTAB. A pH of 6.0 was selected which was closer to the pKa

value of phthalate, is 5.4.

The Peaks of trifluroacetic acid and methane sulfonic acid were

co-eluted to-gather. The increase in the CTAB concentration from

0.25mM to 1.25mM the separation of trifluroacetic acid and methane

sulfonic acid was interchanged. The pH of 6.5 was maximizing the

separation of each organic acid. A good separation was achieved at

the concentration of 1.2mM of CTAB (the critical miscelles

concentration of CTAB is 1.3mM) and pH was 6.5.





Polarity : Negative



Precondition time : 3min (with Hi-flush, at 5bar pressure)

Stop time : 10min.


Buffer : 5mM of Potassium hydrogen

Phthalate and 1.2mM of CTAB

pH : 6.5 with NaOH

5.3.1 Effect of Buffer concentration:

163

The increase in buffer concentration from 2.5mM to 12.5mM,

the resolution was decreased, while peak shape was improved. At

2.5mM of phthalate buffer peaks are broad and a buffer concentration

above 5mM peak shape was good. The run time of analysis was

decreased when the buffer concentration increased. In meantime, the

increased in buffer concentration attributed to high baseline noise. To

compensate the time, resolution, peak shape and a baseline noise an

optimum concentration of 6mM of potassium hydrogen phthalate was

selected for the separation of organic acids.

5.4 Results and Discussion

Organic acids acquire negative charge above the pKa value of

the each acid. The pKa value of formic acid, acetic acid, proponic acid,

trifluroacetic acid, methanesulfonic acid, succinic acid and pivalic

acid are 3.8, 4.8, 4.9, 0.5, -2.0, 4.2 and 5.0 respectively. The pKa2

value of succinic acid is 5.6 and pKa value of potassium hydrogen

phthalate is 5.4. The pKa of all acids are below 6.0, if pH of the buffer

above 6.0, these acids are easily ionizable and acquire a negative

charge in the aqueous solution.

The capillary zone electrophoresis method is a useful tool for

quantifying these organic acids in pharmaceutical drug substances at

low levels. Various buffers of different pH were evaluated for the

effective separation of each organic acid in a single run.

164

The reversing the EOF can reduce the analysis time for anionic

species. This has been used successfully for the analysis of anionic

compounds such as inorganic anions phenolic compounds or

carboxylic acids. A negative power supply causes anionic compounds

to migrate in the same direction as the electro osmotic flow. The

addtion of the cationic surfactants such as hydrophobic quaternary

alkylammoniumm ions, to the buffer, at a concentration below the

critical micellar concentration (CMC), reverse the EOF by forming a

dynamically coating on the inner capillary wall.

Hence a long-chain cationic surfactant, CTAB was added to the

electrolyte to form a positively charged surface coating. The positively

charged surface generates an EOF in the same direction as the

migration of the organic acids, which significantly improves the peak

shapes and reduces the analysis time.

Finally the method was utilized a 6mM of phthalate additive in

the buffer which permits a indirect UV detection and a 1.2mM of

CTAB was added. The pH of the buffer solution was adjusted to 6.5

with sodium hydroxide. A short capillary length, in combination with a

relatively low voltage of (-) 20kV, was selected to give a low current

whilst still maintaining a fast separation time. A low current produces

a flatter baseline and reduces buffer depletion effects. The analyte

signal was set at 330nm and reference signal was set at 210nm, in-

line with the detection of anion probe, which helps to convert a

165

negative peak into positive peak in the electropherogram. The method

was simple, efficient and selective, and has desired level of sensitivity.

The optimized method was validated according to International

Conference on Harmonization (ICH) guidelines38 to prove its

performance characteristics, thereby verifying its suitability and

reliability for monitoring the organic acid in pharmaceutical drug

substances during routine analysis as well as stability studies.

5.4.1 Validation

The following parameters were performed during validation studies,

they are specificity, sensitivity, linearity, precision, stability of sample

solution and accuracy. The data derived from each parameter are

briefly summarized below.

5.4.1.1 Specificty

The solutions of blank, formic acid, acetic acid, trifluoroacetic

acid, methanesulfonic acid, succinic acid, pivalic acid cefazolin

sodium, lamuvidine, lopinavir, and atorvastatin calcium were

introduced into the capillary electrophoresis system to identify the

migration time of each analyte. The migration time of formic acid,

succinic acid, acetic acid, trifluoroacetic acid, methanesulfonic acid,

propionic acid and pivalic acid were 4.1, 4.3, 4.8, 5.2, 5.4, 5.8 and 6.6

min respectively.

166

Organic acids were not detected in the drug substances,

therefore, the each drug substances were spiked with respective

organic acid [Refer Method precision for Level of spiking] along with

other known impurities of each drug substances. While acetic acid in

atorvastatin calcium and succinic acid in lamuvidine were detected

above the limit of quantification of the method, hence these organic

acids were not spiked in the respective drug substances. Each sample

solutions were prepared in triplicate and determined the amount of

respective organic acid present in the each drug substances.

The peaks of each organic acid were well resolved from the

peaks due to blank, other known impurities of the each drug

substances. It indicated that the method was specific for determining

the content of organic acids in drug substances. In conjunction, an

overlay electropherogram of blank solution, organic acids standard

solution, drug substances spiked with desired organic acid along with

other known impurities of drug substances. The specificity values and

method precision values are clubbed together and is given in the table

5.1 to 5.4

Table 5.1 The clubbed data of Specificity and method precision

data for atorvastatin calcium

Atorvastatin Calcium Acetic acid

(%w/w)

Methane sulfonic acid

(%w/w)

Pivalic acid (%w/w)

Method precision-1 0.167 0191 0.095

Method precision-2 0.173 0.195 0.098



167



Specificity-1 0.177 0.204 0.095

Specificity-2 0.182 0.197 0.097

Specificity-3 0.175 0.205 0.104

Average 0.172 0.200 0.100

SD 0.009 0.007 0.004

%RSD 5.2 3.5 4.0

Fig: 5.5a overlay electropherogram of atorvastatin specificity sample

(a) Organic acid standard solution, (b) Atorvastatin sample spiked with all (a) Standard solution of organic acids, (b) Atorvastain calcium sample spiked known related compound of atorvastatin including methanesulfonic acid, acetic acid and Pivalic acid, (c) Blank. Peaks: 1. Formic acid, 2. Succinic acid, 3.Acetic acid, 4. Methanesulfonic acid, 5. Trifluroacetic acid, 6. Propionic acid 7. Pivalic acid

Table 5.2 the clubbed data of Specificity and method precision

data for lopinavir.

Lopinavir Formic acid

(%w/w) Trifluroacetic acid (%w/w)

Methanesulfonic acid (%w/w)





(a)

(b)

(c)

min 0 1 2 3 4 5 6 7 8 9

mAU

0

5

10

15

20 DAD1 B, Sig=330,10 Ref=220,10 (D:\HPCHEM1\1\DATA\SEP-10\ACIMIX58.D)

min* 0 1 2 3 4 5 6 7 8 9

mAU

0

5

10

15

20 *DAD1 B, Sig=330,10 Ref=220,10 (ORG-11\ORGAC011.D)

min* 0 1 2 3 4 5 6 7 8 9

mAU

0

5

10

15

20 *DAD1 B, Sig=330,10 Ref=220,10 (D:\HPCHEM1\1\DATA\SEP-10\ACIMIX77.D)

1 2 3 4

5 6 7

6

6

4

3 7

6

168



Specificity-1 0.049 0.047 0.050

Specificity-2 0.054 0.048 0.049

Specificity-3 0.051 0.048 0.049

Average 0.054 0.049 0.050

SD 0.003 0.002 0.001

%RSD 5.6 4.1 2.0

Fig: 5.5b overlay electropherogram of lopinavir specificity sample

(a) standard solution of Organic acids (b) Lopinavir sample spiked with all known related compound of lopinavir including formic acid, trifluroacetic acid, (c) Blank. Peaks: 1. Formic acid, 2. Succinic acid, 3.Acetic acid, 4. Methanesulfonic acid, 5. Trifluroacetic acid, 6. Propionic acid 7. Pivalic acid

Fig: 5.5c overlay electropherogram of Lamuvidine specificity

sample

(a)

(b)

(c )

min 0 1 2 3 4 5 6 7 8 9

mAU

0 5

10 15 20

DAD1 B, Sig=330,10 Ref=220,10 (D:\HPCHEM1\1\DATA\SEP-10\ACIMIX45.D)

min* 0 1 2 3 4 5 6 7 8 9

mAU

0 5

10 15 20

*DAD1 B, Sig=330,10 Ref=220,10 (D:\HPCHEM1\1\DATA\SEP-10\ACIMIX47.D)

min* 0 1 2 3 4 5 6 7 8 9

mAU

0 5

10 15 20


1

2 3 4 5 6 7

6

3 4

6

1 2 3 4

5 6 7

169

(a) Standard solution of Organic acids, (b) Lamuvidine sample spiked with all known related compound of lamuvidine including Succinic acid and

methanesulfonic acid, (c) Blank. Peaks: 1. Formic acid, 2. Succinic acid,

3.Acetic acid, 4. Methanesulfonic acid, 5. Trifluroacetic acid, 6. Propionic acid 7. Pivalic acid Table 5.3 the clubbed data of Specificity and method precision data for lamuvidine

Lamuvidine Succinic acid (%w/w) Methanesulfonic acid

(%w/w)

Method precision-1 0.546 0.095






Specificity-1 0.507 0.093



Average 0.505 0.095

SD 0.029 0.002

%RSD 5.7 2.1

Fig: 5.5d overlay electropherogram of Cefazolin specificity sample

(a)

(b)

(c )

min 0 1 2 3 4 5 6 7 8 9

0 5

10 15


min* 0 1 2 3 4 5 6 7 8 9

mAU

0 5

10 15 20 25 30


min* 0 1 2 3 4 5 6 7 8 9

mAU

0 2.5

5 7.5 10

12.5 15

17.5 *DAD1 B, Sig=330,10 Ref=220,10 (D:\HPCHEM1\1\DATA\SEP-10\ACIMIX77.D)

1

2 6 1

6

1 4

2 3 5 6 7

170

(a) Standard solution of Organic acids, (b) Cefazolin sample spiked with all

Known related compound of cefazolin including Acetic acid and Pivalic acid, (c) Blank. Peaks: 1. Formic acid, 2. Succinic acid, 3.Acetic acid, 4. Methanesulfonic acid, 5. Trifluroacetic acid, 6. Propionic acid 7. Pivalic acid Table 5.4 the clubbed data of Specificity and method precision data for Cefazolin Sodium

Cefazolin Sodium Acetic acid (%w/w) Pivalic acid (%w/w)










Average 0.051 0.047

SD 0.002 0.001

%RSD 6.5 5.9

(a)

(b)

(c)

min 0 1 2 3 4 5 6 7 8 9

mAU

0

5

10

15


min* 0 1 2 3 4 5 6 7 8 9

mAU

-20

-10

0

10


min* 0 1 2 3 4 5 6 7 8 9

mAU

0 2.5

5 7.5 10

12.5 15

17.5


4 6 7

6

171

5.4.1.2 Sensitivity

The limit of detection (LOD) and limit of quantification (LOQ)

were predicted using slope (S) and residual standard deviation (SD)

that obtained from a linear regression line performed by using organic

acids solution prepared at lower concentration levels between 5µg/mL

and 40µg/mL, is being one of the three approaches described in ICH

guidelines. The formula used for the prediction of LOD and LOQ were

3.3 SD/ S and 10 SD/S respectively.

The slope and standard deviation arrived from the linear regression

lines and therby predicted LOD and LOQ values for formic acid, acetic

acid, succinic acid, trifluroacetic acid, methanesulfonic acid and

pivalic acid were given below

Sl.no Analyte Slope Standard deviation

LOD (µg/mL) LOQ

(µg/mL)

1 Formic acid 0.05370 0.03556 2.19 6.62

2 Acetic acid 0.04228 0.02093 1.63 4.95

3 Succinic acid 0.03595 0.02818 2.59 7.84

4 Trifluroacetic

acid 0.02708 0.01752 2.14 6.47

5 Methanesulfonic

acid 0.03267 0.02301 2.32 7.04

6 Pivalic acid 0.03265 0.01834 1.85 5.62

The solution of oraganic acids were prepared at the predicted

concentration of LOD and LOQ levels, and analyzed for six times.

The percentage relative standard deviation for six replicate

measurements at predicted LOD and LOQ concentration is tabulated

172

in Table 5.5 and 5.6. The represented electropherogram is shown in

the figure 5.2 and 5.3 respectively.

Fig: 5.6 Electropherogram of LOQ solution for organic acids

.

Fig: 5.7 Electropherogram of LOD solution for organic acids

6

7

1 2

3 4

5

173

5

10

5

0

8

7

5

9

6

43

365

3

6

5

5 15

10

mAU ) 17.5

12.5

7.5

5

2.5

0

9

8

7

6

5

4

3 2

1

mi

1

0

2

4

6

1 2 4 7

3 5

Peaks: 1. Formic acid, 2. Succinic acid, 3.Acetic acid, 4. Methanesulfonic acid, 5. Trifluroacetic acid, 6. Propionic acid 7. Pivalic acid

174

Table 5.5. LOQ data of Formic acid, Acetic acid, Succinic acid, Trifluroacetic acid, Methanesulfonic acid

and Pivalic acid

Injection ID Area ratio of Formic acid

Area ratio of Succinic acid

Area ratio of Methane

sulfonic acid Area ratio of Acetic acid

Area ratio of Trifluroacetic

acid Area ratio of Pivalic acid

1 0.38669 0.31592 0.21212 0.25252 0.16029 0.17394

2 0.39381 0.33627 0.21377 0.24313 0.15708 0.15754

3 0.40781 0.33483 0.21430 0.23745 0.14927 0.16257

4 0.39859 0.33957 0.23499 0.24061 0.15726 0.17099

5 0.40602 0.33457 0.21544 0.2552 0.14944 0.16161

6 0.38892 0.31179 0.21801 0.24672 0.15657 0.1694

Mean 0.39697 0.32883 0.21811 0.24594 0.15499 0.17394

SD 0.009 0.012 0.009 0.007 0.005 0.005

% RSD 2.3 3.6 4.1 2.8 3.2 3.2

Concentration (µg/mL)

6.6 7.8 7.0 4.9 6.5 5.6

175

Table 5.6. LOD data of Formic acid, Acetic acid, Succinic acid, Trifluroacetic acid, Methanesulfonic acid

and Pivalic acid.

Injection ID Area ratio of Formic acid

Area ratio of Succinic acid

Area ratio of Methane

sulfonic acid Area ratio of Acetic acid

Area ratio of Trifluroacetic acid

Area ratio of Pivalic acid

1 0.13640 0.11363 0.0700 0.12533 0.0478 0.06946

2 0.12876 0.11091 0.08288 0.12141 0.04733 0.05925

3 0.12843 0.11694 0.07611 0.11671 0.0456 0.05729

4 0.13423 0.11411 0.07485 0.10785 0.0508 0.04857

5 0.12286 0.11375 0.07084 0.10748 0.04973 0.05584

6 0.12532 0.12317 0.06892 0.0947 0.0436 0.06317

Mean 0.12933 0.11542 0.07393 0.11225 0.04748 0.05893

SD 0.005 0.004 0.005 0.011 0.003 0.007

% RSD 3.9 3.5 6.8 9.8 6.3 11.9

Concentration (µg/ml)

2.2 2.6 2.3 1.6 2.1 1.9

176

5.4.1.3 Linearity

The linear relationship of organic acids response against

concentrations were verified in the working concentration range by

analyzing different level of solutions containing each organic acid

from about LOQ level to 40µg/mL. The linear regression lines were

plotted against each organic acid response versus concentration. The

correlation coefficient of the each regression line was found to be more

than 0.99. The statistical analysis of linear regression lines were

evaluated and is summarized in table 5.7 and linearity plot of

concentration of each organic acid Vs area response is shown in the

figure. 5.8

177

Fig: 5.8. Linearity plot of concentration of Organic acids Vs Area

response of organic acids normalized with propionic aicd

0.13

0.63

1.13

1.63

2.13

4.50 9.50 14.50 19.50 24.50 29.50 34.50 39.50

Are

a R

ati

o

Concnetration (ppm)

Linearity Plot Concentration Vs Area Response

Linearity of Formic Acid Linearity of Succinic Acid Linearity of MSA

Linearity of TFA Linearity of Acetic acid Linearity of Pivalic acid

178

Table 5.7 Linearity Data

Concentration Level (ppm)

Formic acid Acetic acid Succinic

acid Trifluroacetic acid

Methanesulphonic acid

Pivalic acid

5 0.29318 0.25359 0.19390 0.13555 0.15474 0.17892

10 0.62225 0.47443 0.41951 0.30169 0.33671 0.38461

15 0.83257 0.65852 0.55617 0.40886 0.47373 0.51108

20 1.10711 0.87892 0.74536 0.55479 0.6291 0.68054

25 1.31252 1.03205 0.90119 0.6556 0.75156 0.80943

30 1.59111 1.28393 1.07494 0.78714 0.9386 0.98469

35 1.89302 1.47965 1.24879 0.93518 1.1035 1.14735

40 2.18544 1.71053 1.49602 1.0839 1.29866 1.31483

Slope 0.0537 0.04228 0.03595 0.02708 0.03267 0.03265

Intercept 0.047 0.045 0.027 0.014 -0.005 0.035

STEYX 0.03556 0.02093 0.02818 0.01752 0.02301 0.01834

CC 0.9987 0.99926 0.99822 0.99873 0.99851 0.99904

RSQ 0.9974 0.9985 0.9964 0.9975 0.9970 0.9981

LOD 2.19 1.63 2.59 2.14 2.32 1.85

LOQ 6.62 4.95 7.84 6.47 7.04 5.62

179

5.4.1.4 Precision

5.4.1.4a System Precision

The system precision was demonstrated by performing six

replicate injection of organic acid standard solution (25g/mL) into

capillary electrophoresis system and the percentage relative standard

deviation of response for six replicate measurements was found to be

less than 5.0 for each organic acid and standard electropherogram is

shown in figure 5.9.

5.4.1.4b Method precision

Repeatability of the test method (method precision) was

demonstrated by analyzing six separate sample solution prepared

using single batch of cefazolin sodium sample spiked with each

0.05%w/w of acetic acid and pivalic acid, lopinavir drug substance

was spiked with formic acid, trifluoroacetic acid and methanesulfonic

acid at about 0.05%w/w. The lamivudine drug substance was spiked

with methanesulfonic acid at about 0.1%w/w and atorvatatin calcium

drug substance was spiked with mthanesulfonic acid at about

0.2%w/w and pivalic acid at about 0.1%w/w. Since these organic

acids were not detected in as such samples. However succinic acid

and acetic acid were detected in sample well above the LOQ level in

lamivudine and atorvastatin calcium drug substances respectively.

The content of each organic acid in each drug substance was

180

determined. The percentage relative standard deviations for six

replicate measurements are tabulated in table 5.8 to 5.12.

5.4.1.4c Intermediate precision

Intermediate precision of the method was performed in the same way

as described in method precision, however, by employing different

analyst on other day using another lot of capillary. The content of

organic acid was determined in each preparation, and the percentage

relative standard deviation for six replicate measurements was found

to be less than 5.0. The percentage relative standard deviations for six

replicate measurements are tabulated in table 5.8 to 5.12.

Fig: 5.9 Electropherogram of standard organic acids

Peaks: 1. Formic acid, 2. Succinic acid, 3.Acetic acid, 4. Methanesulfonic acid, 5. Trifluroacetic acid, 6. Propionic acid 7. Pivalic acid

.

1 4

2 3

6

5 7

mAU

181

Table 5.8 Precision Data for Lopinavir

Sample Formic acid (%w/w)

Trifluroacetic acid(%w/w)


Set I Set II Set I Set II Set I Set II

1 0.055 0.052 0.052 0.056 0.051 0.048

2 0.059 0.054 0.052 0.055 0.050 0.046

3 0.055 0.053 0.048 0.056 0.051 0.046

4 0.055 0.055 0.050 0.055 0.052 0.046

5 0.052 0.059 0.047 0.057 0.051 0.045

6 0.057 0.054 0.045 0.054 0.050 0.049

Mean 0.056 0.055 0.049 0.056 0.051 0.046

SD 0.002 0.003 0.003 0.001 0.001 0.001

%RSD 3.6 5.5 6.1 1.8 2.0 2.2

95% CI 0.002 0.003 0.003 0.001 0.001 0.001

Overall mean

0.055 0.052 0.049

Overall SD 0.002 0.004 0.002

Overall RSD (%)

3.6 7.7 4.1

Table 5.9 Precision Data for Cefazolin sodium

Sample Acetic acid (%w/w) Pivalic acid (%w/w)

Set I Set II Set I Set II

1 0.050 0.054 0.049 0.049

2 0.051 0.054 0.047 0.049

3 0.049 0.051 0.045 0.048

4 0.050 0.053 0.047 0.050

5 0.051 0.053 0.047 0.048

6 0.050 0.051 0.046 0.049

Mean 0.050 0.053 0.047 0.049

SD 0.001 0.001 0.001 0.001

%RSD 3.3 3.0 5.9 5.3

95% CI 0.001 0.001 0.001 0.001

Overall mean 0.051 0.048

Overall SD 0.002 0.001

Overall RSD (%) 6.5 5.6

182

Table 5.10 Precision Data for Atorvastatin Calcium

Sample Acetic acid (%w/w)


Pivalic acid (%w/w)

Set I Set II Set I Set II Set I Set II

1 0.167 0.174 0.191 0.189 0.095 0.098

2 0.173 0.174 0.195 0.182 0.098 0.099

3 0.158 0.170 0.192 0.184 0.101 0.103

4 0.170 0.164 0.209 0.188 0.106 0.101

5 0.164 0.163 0.206 0.185 0.104 0.097

6 0.185 0.161 0.197 0.185 0.098 0.106

Mean 0.170 0.168 0.198 0.186 0.100 0.101

SD 0.009 0.006 0.007 0.003 0.004 0.003

%RSD 5.3 3.6 3.5 1.6 4.0 3.0

95% CI 0.007 0.006 0.009 0.003 0.004 0.003

Overall mean 0.169 0.192 0.101

Overall SD 0.007 0.009 0.004

Overall RSD (%) 4.1 4.7 4.0

Table 5.11 Precision Data for Lamuvidine

Sample

Succinic acid (%w/w) Methanesulfonic acid (%w/w)

Set I Set II Set I Set II

1 0.323 0.323 0.095 0.092

2 0.298 0.299 0.093 0.086

3 0.322 0.301 0.098 0.092

4 0.323 0.311 0.098 0.092

5 0.325 0.298 0.094 0.091

6 0.325 0.331 0.096 0.094

Mean 0.318 0.311 0.096 0.091

SD 0.011 0.014 0.002 0.003

%RSD 3.5 4.5 2.1 3.3

95% CI 0.013 0.016 0.002 0.003

Overall mean

0.315 0.093

Overall SD 0.013 0.003

Overall RSD (%)

4.1 3.2

183

Table 5.12 System precision

Area ratio of Propionic acid with

Injection Formic acid Acetic acid Succinic acid Trifluroacetic acid Pivalic acid

Injection-1 1.23362 0.82724 1.11275 0.75816 0.57854

Injection-2 1.20986 0.82540 1.09198 0.72401 0.60256

Injection-3 1.20765 0.80204 1.13311 0.77921 0.58434

Injection-4 1.21993 0.82832 1.04269 0.81360 0.62681

Injection-5 1.18532 0.80506 1.00786 0.75324 0.60926

Injection-6 1.18063 0.84514 1.13397 0.79266 0.59379

Mean 1.20617 0.8222 1.08706 0.77015 0.59922

SD 0.020 0.016 0.051 0.032 0.018

%RSD 1.7 1.9 4.7 4.2 3.0

184

5.4.1.5 Accuracy

The accuracy of the method was verified by preparing sample

solution spiked with known amount of organic acid at three different

concentration levels. The pivalic acid and acetic acid in cefazolin

sodium and trifluoroacetic acid, methanesulfonic acid and formic acid

in lopinavir were spiked at about LOQ, 0.05% and 0.075%w/w level.

Similarly in lamivudine sample was spiked with succinic acid and

methanesulfonic acid at about 0.05%, 0.1% and 0.15%w/w. The

atorvastatin calcium sample was spiked with acetic acid at about

0.15%, 0.3% and 0.45%w/w, methanesulfonic acid was at about

0.1%, 0.2% and 0.3%w/w and pivalic acid at about 0.05%, 0.1% and

0.15%w/w. Each concentration level was prepared in triplicate and

analyzed as per the method. The percentage recovery of the each

analyte was evaluated and it was lies between 85% and 114%. The

results are tabulated in table 5.13 to 5.16. These data reveals that

this method is accurate for determination of organic acids in various

pharmaceutical drug substances.

185

Table 5.13 Recovery table for Formic acid, Methane sulfonic acid and Trifluroacetic acid in Lopinavir

Concentration/ Sample ID

Amount added (%w/w) Amount Recovered

(%w/w) % Recovery Statistical Analysis

Formic

acid MSA TFA

Formic acid

MSA TFA Formic

acid MSA TFA

Formic acid

MSA TFA

Sample 1

Nil Nil Nil -- -- -- -- -- --

Mean SD %RSD

Sample 2

-- -- -- Sample 3

50%-Sample 1 0.016 0.020 0.029 0.018 0.017 0.025 112.5 85.0 86.2 110.4 88.3 94.3

50%-Sample 2 0.016 0.020 0.029 0.017 0.019 0.031 106.3 95.0 106.9 3.58 5.77 11.08

50%-Sample 3 0.016 0.020 0.029 0.018 0.017 0.026 112.5 85.0 89.7 3.2 6.5 11.7

100%-Sample 1 0.051 0.050 0.052 0.054 0.047 0.051 105.9 94.0 98.1 108.5 89.3 91.7

100%-Sample 2 0.051 0.050 0.052 0.058 0.043 0.046 113.7 86.0 88.5 4.50 4.16 5.54

100%-Sample 3 0.051 0.050 0.052 0.054 0.044 0.046 105.9 88.0 88.5 4.1 4.7 6.0

150%-Sample 1 0.076 0.075 0.078 0.075 0.069 0.073 98.7 92.0 93.6 103.1 96.9 94.9

150%-Sample 2 0.076 0.075 0.078 0.077 0.077 0.076 101.3 102.7 97.4 5.47 5.41 2.19

150%-Sample 3 0.076 0.075 0.078 0.083 0.072 0.073 109.2 96.0 93.6 5.3 5.6 2.3

Overall Statistical Analysis

Mean SD % RSD Mean SD % RSD

Formic acid 107.3 5.166 4.8 Methane sulfonic

acid 91.5 6.036 6.6

Trifluroacetic

acid 93.6 6.457 6.9

186

Table 5.14 Recovery table for Acetic acid, and Pivalic acid in cefazolin Sodium

Concentration/ Sample ID

Amount added Amount Recovered % Recovery

Statistical Analysis Acetic acid

(%w/w)

Pivalic acid

(%w/w)

Acetic acid

(%w/w)

Pivalic acid

(%w/w)

Acetic acid

(%w/w)

Pivalic acid

(%w/w)

Sample 1

Nil

ND ND

-- -- Acetic acid

(%w/w)

Pivalic acid

(%w/w) Sample 2 ND ND

Sample 3 ND ND

50%-Sample 1 0.028 0.026 0.029 0.025 103.6 96.2 Mean 103.5 101.2

50%-Sample 2 0.028 0.026 0.029 0.027 103.6 103.8 SD 0.231 4.331

50%-Sample 3 0.031 0.028 0.032 0.029 103.2 103.6 % RSD 0.2 4.3

100%-Sample 1 0.050 0.046 0.051 0.048 102 104.3 Mean 101.3 97.1

100%-Sample 2 0.050 0.046 0.049 0.043 98 93.5 SD 3.012 6.207

100%-Sample 3 0.051 0.047 0.053 0.044 103.9 93.6 % RSD 3.0 6.4

150%-Sample 1 0.071 0.075 0.071 0.078 100 104.6 Mean 101.4 101

150%-Sample 2 0.071 0.076 0.073 0.075 102.8 98.5 SD 1.4 3.179

150%-Sample 3 0.072 0.076 0.073 0.076 101.4 100 % RSD 1.4 3.1


Mean SD % RSD

Acetic acid 102.1 1.973 1.9

Pivalic acid 99.8 4.563 4.6

ND: Not detected

187

Table 5.15 Recovery table for Succinic acid, and Methanesulphonic acid in Lamuvidine

Concentration/

Sample ID

Amount added

Amount Recovered %

Recovery

Statistical Analysis

MSA (%w/w) Succinic

acid MSA

(%w/w) Succinic

acid MSA

(%w/w) Succinic

acid

Sample 1

Nil

ND 0.007

-- -- MSA

(%w/w) Succinic

acid Sample 2 ND 0.008

Sample 3 ND 0.011

50%-Sample 1 0.050 0.250 0.050 0.234 100.00 93.56 Mean 96.7 90.9

50%-Sample 2 0.050 0.249 0.048 0.224 96.00 89.91 SD 3.06 2.365

50%-Sample 3 0.050 0.251 0.047 0.224 94.00 89.13 % RSD 3.2 2.6

100%-Sample 1 0.100 0.502 0.095 0.538 95.00 107.17 Mean 93.3 104.0

100%-Sample 2 0.100 0.499 0.093 0.524 93.00 104.96 SD 1.53 3.777

100%-Sample 3 0.100 0.499 0.092 0.498 92.00 99.81 % RSD 1.6 3.6

150%-Sample 1 0.150 0.747 0.141 0.732 94.00 98.02 Mean 94.7 95.5

150%-Sample 2 0.150 0.746 0.147 0.691 98.00 92.63 SD 3.06 2.711

150%-Sample 3 0.150 0.747 0.138 0.716 92.00 95.84 % RSD 3.2 2.8


Mean SD % RSD

Succinic acid 96.8 6.322 6.5

Methane sulfonic acid 94.9 2.713 2.9

188

Table 5.16 Recovery table for Formic acid, Methane sulfonic acid and Pivalic acid in Atorvastatin

Calcium Concentration/ Sample ID

Amount added (%w/w) Amount Recovered

(%w/w) % Recovery Statistical Analysis

Acetic acid

MSA Pivalic acid

Acetic acid

MSA Pivalic acid

Acetic acid

MSA Pivalic acid

Acetic acid

MSA Pivalic acid

Sample 1 Nil Nil Nil 0.173 Nil Nil -- -- --



50%-Sample 1 0.149 0.105 0.052 0.157 0.094 0.049 105.3 90.0 94.2 Mean 102.4 94.0 92.9

50%-Sample 2 0.148 0.104 0.051 0.153 0.105 0.048 103.1 101.0 94.1 SD 3.25 6.06 2.17

50%-Sample 3 0.149 0.104 0.052 0.147 0.095 0.047 98.9 91.1 90.4 %RSD 3.2 6.4 2.3

100%-Sample 1 0.298 0.209 0.102 0.312 0.197 0.101 104.6 94.3 99 Mean 102.9 92.9 99

100%-Sample 2 0.298 0.209 0.102 0.300 0.193 0.099 100.8 92.6 97.1 SD 1.94 1.23 1.95

100%-Sample 3 0.297 0.208 0.101 0.307 0.191 0.102 103.4 91.9 101 %RSD 1.9 1.3 2

150%-Sample 1 0.446 0.313 0.151 0.442 0.290 0.146 99.0 92.8 96.7 Mean 99.8 91.3 98.3

150%-Sample 2 0.447 0.313 0.152 0.445 0.283 0.148 99.5 90.5 97.4 SD 1.04 1.33 2.14

150%-Sample 3 0.446 0.312 0.151 0.450 0.282 0.152 101.0 90.5 100.7 %RSD 1.0 1.5 2.2


Mean SD % RSD Mean SD % RSD

Acetic acid 101.7 2.436 2.4 Methane sulfonic acid 92.7 3.384 3.7

Pivalic acid 97.0 3.412 3.5

189

5.4.1.6 Robustness

Robustness of the method was verified by deliberately altering

the critical method parameters from that of actual conditions. The

altered conditions include change in temperature, buffer pH, buffer

concentration, precondition time, injection time and applied voltage.

The results obtained from robustness experiments indicated that, the

method parameters were suitably optimized to tolerate minor

variations in the method. The data for resolution between peaks is

shown in table 5.17.

190

Table 5.17 Robustness data for variable parameter.

Resolution

Sl.No

Method Variable Parameter

Formic acid

Succinic acid

Methane sulfonic

acid

Acetic acid Trifluroacet

ic acid Proponic

acid Pivalic acid

1 As such Method condtion

- 1.94 6.49 5.60 2.31 3.41 7.38

2 pH 6.3 - 3.26 4.82 6.80 1.12 5.43 8.18

3 pH 6.7 - 1.99 6.92 5.93 2.17 3.17 7.92

4 Conc. CTAB 1.15mmole - 2.65 6.04 6.53 1.71 5.12 8.09

5 Conc. CTAB 1.25mmole - 2.90 5.73 6.35 1.63 5.42 8.18

6 Conc. of Buffer 5mmole - 2.50 5.56 6.44 0.98 5.19 7.58

7 Conc. of Buffer 7mmole - 2.92 6.35 6.80 1.25 5.39 8.49

8 Voltage -18kV - 2.44 6.45 6.04 1.98 4.17 8.11

9 Voltage -22kV - 2.08 6.13 5.43 1.97 3.45 7.09

10 Temperature 18°C - 2.50 6.33 6.35 1.97 4.10 7.89

11 Temperature 22°C - 2.19 6.02 5.77 1.94 4.25 7.46

12 Precodition Time 4min - 2.35 6.09 6.04 1.93 4.01 7.70

13 Precodition Time 2min - 2.35 6.07 5.99 2.05 5.24 7.92

12 Injection Time 4min - 2.47 6.61 6.16 1.96 4.09 7.91

13 Injection Time 6min - 2.31 5.99 5.94 1.87 4.59 7.55

191

5.5 Conclusion

The capillary electrophoresis method to determine the content of

organic acid in various pharmaceutical drug substances is optimized

with the background electrolyte of Potassium hydrogen phthalate and

CTAB. This method was validated according to the ICH guildlines and

summarized validation data is presented in the table 5.18.

192

Table 5.18. Summary of Validation Data.

Validation parameter

Results

Formic acid Succinic

acid

Methane sulfonic

acid Acetic acid

Trifluoro acetic acid

Pivalic acid

Correlation coefficient (r2) 0.9987 0.9982 0.9985 0.9993 0.9987 0.9990

Concentration range (g/ml) Between 5 and 40

Intercept 0.047 0.027 -0.005 0.045 0.014 0.035

Slope 0.0537 0.03595 0.03267 0.04228 0.02708 0.03265

Standard error estimate (Residual standard deviation)

0.03556

0.02818

0.02301

0.02093

0.01752 0.01834

System Precision (%RSD) 2.1 1.7 2.2 3.5 3.7 3.5

Method Precision (%RSD) 3.6 4.0 3.6 5.4 6.1 5.9

Intermediate Precision (%RSD) 4.8 3.1 3.5 3.4 2.1 5.3

Limit of detection (g/ml) 2.2 2.6 2.3 1.6 2.1 1.9

Limit of detection Precision %RSD

3.9 3.5 6.8 9.8 6.3 11.9

Limit of quantification (g/ml) 6.6 7.8 7.0 4.9 6.5 5.6

Limit of quantification Precision %RSD

2.3 3.6 4.1 2.8 3.2 3.6

Overall % Recovery 106.6 102.0 95.1 102.0 93.0 99.9

Overall % RSD 3.2 1.8 1.9 1.2 2.0 2.5

191

5.6 References

1 Villers, A. D., Lyen, F., Crouch, A., A robust capillary

electrophoresis method for the determination of organic acids in

wine, Eur.Food.Res.Technol., 271, 2003, 535-540.

2 Mato, I., Huidobro, J. F., Simal-Lozano, J., Sancho, M. T.,

Simultaneous determination of organic acids in beverages by

capillary zone electrophoresis, Anal.Chim.Acta, 565, 2006, 190-

197.

3 Cortacero-Ramirez, S., Segura-Carretero, A., Hernainz-Bermudez

de Castro, M., Fernandez-Gutierrez, A., Determination of low-

molecular-mass organic acids in any type of beer samples by co-

electroosmotic capillary electrophoresis, J. Chromatogr. A, 1064,

2005, 115-119.

4 Kelly, L., Nelson, R. J., Capillary electrophoresis of organic acids

and anions, J. Liq. Chromatogr., 16, 1993, 2103.

5 Tindal, G. W., Wilder, D. R., Perry, R. L., Optimizing dynamic

range for the analysis of small ions by capillary electrophoresis

(aliphatic acid indirect UV), J. Chromatogr. A, 641, 1993, 163-

167.

6 Shirao, M., Furuta, R., Suzuki, S., Nakazwa, H., Fujita, S.,

Maruyama, T., Determination of organic acids in urine by

capillary zone electrophoresis, J. Chromatogr. A, 716, 1994, 247-

251.

7 Mato, I., Suarez-Luque, S., Huidobro, J. F., Simple determination

of main organic acids in grape juice and wine by using capillary

192

zone electrophoresis with direct UV detection, Food Chem., 102,

2007, 104-112.

8 Altria, K. D., Assi, K. H., Bryant, S. M., and Clark B. J.,

Determination of organic acid drug counter-ions by capillary

electrophoresis, Chromatographia, 44, 1997, 367-371.

9 Altria, K. D., Frake, P., Gill, I., Hadgatt, T., Kelly, M. H., Rudd, D.

R., Validated capillary electrophoresis method for the assay of a

range of basic drugs, J. Phar. Biomed.Anal., 13, 1995, 951-957.

10 Altria, K. D. Wood, T., Kitscha, R., Intosh, A. R., Validation of a

capillary electrophoresis method for the determination of

potassium counter –ion levels in an acidic drug salt, J. Phar.

Biomed.Anal., 13, 1995, 33-38.

11 Altria, K. D., Fabre, H., Approaches to optimization of precision in

capillary electrophoresis, Chromatographia, 40, 1995, 313-320.

12 Altria, K. D., Goodall, D. M., Rogan, M. M., Quantitative

determination of drug counter ion stoichiometry by capillary

electrophoresis, Chromatogrphia, 38, 1994, 637-642.

13 Volgger, D., Zemann, A.J., Bonn, G. K., Antal, M. J., high- speed

separation of carboxylic acids by co-electro osmotic capillary

electrophoresis with direct and indirect UV detection, J.

Chromatogr. A, 758, 1997, 263-276.

14 Arellano, M., Andrianary, J., Dedieu, F., Conderc, F. Puig, P.,

Method development and validation for the simultaneous

determination of organic acid and inorganic acids by capillary

zone electrophoresis, J. Chromatogr. A, 765, 1997, 321-328.

193

15 Zemann, A. J., Sub minute separation of organic acid and

inorganic anions with co-electroosmotic capillary electrophoresis,

J. Chromatogr. A, 787, 1997, 243-251.

16 Wiedmer, S. K., Siren, H., Riekkula, M. L., Jumppanem, J. H.,

Comparison of capillary electrophoresis system for the reliable

identification of some carboxylic acids using the marker

technique, J. Chromatogr. A, 781, 1997, 239-249.

17 Rivasseu, C., Boisson, A., Mongelard, G., Couram, G., Bastien, O.,

Bligny, R., Rapid analysis of organic acids in plant extract by

capillary electrophoresis with indirect UV detection: Direct

metabolic analyses during metal stress, J. Chromatogr. A, 1129,

2006, 283-290.

18 Rovio, S., Kolliola, A., Siren, H., Tamminen, T., Determination of

the carboxylic acids in acidic and basis process samples by

capillary zone electrophoresis, J. Chromatogr. A, 1217, 2010,

1407-1413.

19 Noblitt, S. D., Mazzoleni, L. R., Hering, S. V., Collett Jr. J. L.,

Henry, C. S., Separation of common organic and inorganic anions

in atmospheric aerosols using a piperazine buffer and capillary

electrophoresis, J. Chromatogr. A, 1154, 2007, 400-406.

20 Bylund, D., Norstrom, S. H., Essen, S. A., Lundrrom, U. S.,

Analysis of low molecular mass organic acids in natural waters by

ion exclusion chromatography tandem mass spectrometry, J.

Chromatogr. A, 1176, 2007, 89-93.

194

21 Geng, X., Zhang, S., Wang, Q., Zhao, Z., Determination of organic

acids in presence of inorganic anions by ion chromatography with

suppressed conductivity detection, J. Chromatogr. A, 1192, 2008,

187-190.

22 Suzuki, Y., Automated analysis of low molecular weight organic

acids in ambient air by a microporous tube diffusion scrubber

system coupled to ion chromatography, Anal. Chim Acta, 353,

1997, 227-237.

23 Kuo, C., Improved application of ion chromatographic

determination of carboxylic acids in ozonated drinking water, J.

Chromatogr. A, 804, 1998, 265-272.

24 Chen, Z. L., Gold, B. K., Adams, M. A., Indirect photometric

detection of aliphatic acids by ion exclusion chromatography

using aromatic acid eluents, J.Chromatogra. A, 818, 1998, 61-68.

25 Caliamanis, A., McCormick, M. J., Carpeter, P. D.,

Conductometric detection of anions of weak acids in chemically

suppressed Ion chromatography, Anal. Chem., 69 1997, 3272-

3276.

26 Marales, M. L., Gonazales, A. G., Troncoso, A. M., Ion-exclusion

chromatographic determination of organic acids in vinegars, J.

Chromatogr. A, 822, 1997, 45-51.

27 Levart, A., Gucek, M., Pihlar, B., Veber, M., Determination of

Organic acids in Air by capillary electrophoresis and Ion exclusion

chromatography, Chromatographia, 51, 2000, S-321-324.

195

28 Kaniansky, D., Masar, M., Marak, J., Bodor, R., Capillary

electrophoresis of inorganic anions, J. Chromatogr. A, 836, 1999,

133-178.

29 Farre, J. Borrull, F. Callul, M. Application of capillary

electrophoresis in the quality control of osmotically treated water,

Chromatographia, 44, 1997, 235-239.

30 Souza, S. R., Tavares, M. F. M., DeCarvalho, L. R. F., Systematic

approach to the separation of mono- and hydroxycarboxylic acids

in environmental samples by ion chromatography and capillary

electrophoresis, J. Chromatogr. A, 796, 1996, 335-346.

31 Mainka, A., Ebert, P., Kibler, M., Prokop, T., Tenberken, B.,

Bachmann, K., Development of new methods for the analysis of

carboxylic acids and carbonyl compounds in size classified

raindrops by CE for application in modeling atmospheric process.,

Chromatographia, 45, 1997, 158-162.

32 Soga, T., Ross, G. A Capillary electrophoretic determination of

inorganic and organic anions using 2,6-pyridinedicarboxylic acid:

effect of electrolyte complexing ability, J. Chromatogr. A, 767,

1997, 223-230.

33 International conference on harmonization of technical

requirements for registration of pharmaceuticals for human use,

ICH harmonized tripartite guideline. Impurities in new drug

substances Q3A (R2), step 4 2006.

34 http://en.wikipedia.org/wiki/Atorvastatin.

35 http://en.wikipedia.org/wiki/Lopinavir.

196

36 http://en.wikipedia.org/wiki/Lamivudine.

37 http://en.wikipedia.org/wiki/Cefazolin.

38 International conference on harmonization of technical

requirements for registration of pharmaceuticals for human use,

ICH harmonized tripartite guideline. Validation of Analytical

procedures Text and methodology Q2 (R1), step 4 2005.

CHAPTER-5shodhganga.inflibnet.ac.in/bitstream/10603/4552/14/14_chapter 5.pdf · Cefamezin,...

Documents

Transcript of CHAPTER-5shodhganga.inflibnet.ac.in/bitstream/10603/4552/14/14_chapter 5.pdf · Cefamezin,...