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Sensitive ion chromatographic determination of citrate and formate in

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 Vol.4, No.4 (2011), 844-852
ISSN: 0974-1496
CODEN: RJCABP
http://www.rasayanjournal.com

SENSITIVE ION CHROMATOGRAPHIC DETERMINATION OF

CITRATE AND FORMATE IN PHARMACEUTICALS
T.Kaleemullah1*, Mansur Ahmed 1, Hemant Kumar Sharma2
and M. Nageswara Rao2
1
Post Graduate and Research Department of Chemistry, Islamiah College (Affiliated to
Thiruvalluvar University), Vaniyambadi-635752, Vellore Dist, Tamil Nadu, India
2
Aurobindo Pharma Limited Research Centre, 313, Bachupally,
Hyderabad-500 090, Andhra Pradesh, India
*E-mail: kaleem78@rediffmail.com

ABSTRACT
A simple and sensitive Ion chromatographic method has been developed for the determination of sodium citrate and
formic acid in Penicillin class of drugs. Efficient retention was achieved in Hamilton PRP-X 300, column 250mm
long with 4.0mm I.D., 7µm particle diameter with conductometric detection. The mobile phase delivered in an
isocratic mode at a flow rate of 1.0 ml min-1 at ambient temperature. The developed method was optimized and
validated for its linearity, accuracy, robustness, and precision. The concentration range for linearity experiment was
10.8 µg mL-1 to 64.7µg mL-1 for sodium citrate and 1.5µg mL-1 to 15.0µg mL-1 for formic acid respectively. The
limit of quantification and detection for formic acid was found to be 1.2µg mL-1 and 0.4µg mL-1 respectively.
Keywords: Ion chromatography, Nafcillin for injection, Penicillin G potassium for injection, Ampicillin Trihydrate,
Sodium citrate, Formic acid.
© 2011 RASĀYAN. All rights reserved.

INTRODUCTION
Trisodium citrate is widely used in many pharmaceutical preparations as flavoring and stabilizing agent.
Chemically it is Trisodium 2-hydroxypropane-1,2,3-tricarboxylate referred as sodium citrate, having
chemical formula of Na3C6H5O7 shown in Figure 1(a). Sodium citrate is used to relieve discomfort in
urinary tract infections, such as cystitis, to reduce the acidosis in distal renal tubular acidosis1. Sodium
citrate has been used as an anticoagulant to stabilize blood2. ß-lactam antibiotics containing sodium citrate
as buffer additives in their preparation has been selected.
Nafcillin sodium is an narrow spectrum, ß-lactamase resistant antibiotic3-4, chemically, it is Monosodium
(2S,5R,6R)-6-(2-ethoxy-1-naphthamido)-3,3-dimethyl-7-oxo-4-thia-1-aza bicyclo [3.2.0]-heptane-2-
carboxylate monohydrate shown in Figure 1(b). Nafcillin for injection containing about 35 mg per gram
of trisodium citrate for the optimization of drug pH intended for intravenous or intramuscular
administration5.
Penicillin G potassium is ß-lactamase sensitive antibiotic, its pharmaceutical preparation is a sterile dry
mixture of Penicillin G potassium with sodium citrate of 4 to 5% addition to optimize the pH6.
Chemically, it is Monopotassium 3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1-azabicyclo[3.2.0]-
heptane-2-carboxylate shown in Figure 1(c). Various analytical method has been reported for the
quantification of sodium citrate or citric acid by potentiometric7, colorimetric8, photometric9, high
performance liquid chromatography10, 11, anion exchange12, and ion exclusion method13. In general ion-
exclusion requires longer run time for citric acid if organic modifiers are not used. Ion chromatography
using indirect photometric detection of citrate assay has been described by Chalgari and Tan14, utilizes
low mobile phase pH to ionize the citric acid, with strong UV absorbing chromophore because citrate ion
is poorly absorbing analyte. The method requires proper pH adjustment for the control of citrate
ionization to obtain consistent retention time15.

STUDY OF CITRATE AND FORMATE T.Kaleemullah et al.

 Vol.4, No.4 (2011), 844-852

Formic acid is an important intermediate in chemical synthesis having chemical formula HCO2H. Formic
acid is used as a source for formyl group during formylation reaction16. Ampicillin Trihydrate is an
extended spectrum ß-lactam antibiotic, chemically it is (2S,5R,6R)-6-[[(2R)-2-amino-2-
phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]-heptane-2-carboxylic acid
Trihydrate shown in Figure 1(d). The signal due to small organic moiety will largely suppress by the
elution of drug substance by direct UV detection, quantification of such ion becomes difficult in bulk
matrix. Various analytical methods are available for the determination of formic acid by colorimetric17,
monometric18, capillary electorphorosis19-20, HPLC21, GC/MS22, NMR spectrometry23, ionic HPLC24 and
ion chromatographic method25-26. Therefore a rapid, economical, selective and simple ion
chromatographic method has been proposed for the determination of sodium citrate as citrate in dosage
forms and formic acid as formate in drug substance.

EXPERIMENTAL
Reagents and Chemicals
Reference standard of Sodium citrate tribasic dihydrate and Concentrated Sulfuric acid procured from
Merck Limited, Mumbai, India. Highly purified water obtained from Milli-Q purification system and
Rankem (RFCL), India. Formic acid purchased from Merck KGaA, Darmstadt, Germany. The
investigation samples were procured from Aurobindo Pharma Ltd, Hyderabad, India.

Preparation of standard solutions

Sodium citrate standard solution
Accurately weighed 50 mg of Sodium citrate tribasic dihydrate was transferred to a 50 mL volumetric
flask, added about 30 mL of diluent and sonicated to dissolve, diluted up to the make with diluent and
mixed. Further, 1 mL of the resulting solution was diluted to 20 mL with diluent and filtered the solution
through 0.45µm Millex HV PVDF membrane filter. This solution contains 0.05mg/mL.
Formic acid standard solution
Accurately weighed 50 mg of Formic acid was transferred to a 50 mL volumetric flask, added about 30
mL of diluent and sonicated to dissolve, diluted up to the make with diluent and mixed. Further, 2 mL of
the resulting solution was diluted in 20 mL with diluent and further 1.5 mL of the solution was diluted to
50 mL with diluent and filtered the solution through 0.45µm Millex HV PVDF membrane filter. This
solution contains 0.003mg/mL.

Preparation of sample solutions

Sodium citrate sample solution
Accurately weighed 125 mg of Formic acid was transferred to a 100 mL volumetric flask, added about 70
mL of diluent and sonicated to dissolve, diluted up to the make with diluent and filtered the solution
through 0.45µm Millex HV PVDF membrane filter.
Formic acid sample solution
Accurately weighed 50 mg of Formic acid was transferred to a 20 mL volumetric flask, added about 15
mL of diluent and sonicated to dissolve, diluted up to the make with diluent and filtered the solution
through 0.45µm Millex HV PVDF membrane filter.

Equipments and conditions

An ion chromatography system Metrohm 733 separation centre equipped with 732 conductometric
detector, Metrohm 709 IC pump, suppressor with peristaltic pump, Metrohm 813 compact auto sampler
and 762 IC interface with Metrohm IC net 2.3 data handling system was used. A 250mm long and 4.6mm
internal diameter having 7µm particle diameter column packed with polystyrene divinyl benzene
copolymer having sulfonic acid group (Make: Hamilton PRP-X 300) has been selected. Mobile phase was
0.05mM Sulfuric acid buffer and flow rate was set to 1.0 ml min-1 and injection volume set to 20µL with
data acquisition time of 30 minutes. Purified Milli-Q water was used as diluent

STUDY OF CITRATE AND FORMATE 845 T.Kaleemullah et al.

 Vol.4, No.4 (2011), 844-852

RESULTS AND DISCUSSION

Validation of the method
The proposed IC method has been validated as per guideline27 for specificity, linearity, accuracy, limit of
detection and limit of quantification, precision and stability of sample solution. Validation activity has
been divided in to two parts, (I) Sodium citrate content in Nafcillin for injection and Penicillin G
potassium for injection uses assay procedure while (II) Formic acid in Ampicillin Trihydrate drug
substance follows content procedure.

NaO O
H2O H
OH O N
O H3 C CH3
O O .H2O
- + CH3
+ - O Na NH S
Na O H
- + H
O O Na

(a). Trisodium Citrate (b). Nafcillin Sodium Monohydrate

H COOK H COOH
HO CH3
O
CH3 H NH2 N
N . 3H2O
NH CH3 NH
S S CH3
O H H H H
O
(c). Penicillin G potassium (d). Ampicillin Trihydrate

Fig.-1: Chemical structures of (a).Trisodium citrate, (b). Nafcillin sodium monohydrate, (c).Penicillin G potassium,
and (d). Ampicillin Trihydrate

Validation of trisodium sodium citrate as citric acid

Selectivity
The sample solution of impurities and standard was prepared based on the concentration of selected
pharmaceuticals individually for Nafcillin for injection (NFI), Penicillin G potassium for injection (PFI),
has been injected into the chromatographic system individually to identify the retention time. The
retention time of Citrate was found to be about 3.5 min. However, the known related impurities and
concentrated main peak of the respective drugs were not eluting with the analyte peak. These samples
were found to contain 4 to 5% of sodium citrate moiety in their formulations. Therefore, these samples
were spiked with known related substances at about 0.1% w/w level without sodium citrate as well as
with sodium citrate. From the above verification, representative chromatogram obtained from (a) Sodium
citrate standard, (b) PFI sample spiked with its related substance, (c) NFI sample spiked with its related
substance, and (d) Diluent were shown in Figure 2. The overall %RSD was found to be very much within
the acceptance criteria (Less than 10%) for the investigation samples, thereby indicating that the method
is selective for determining the content of citrate in pharmaceuticals individually.
Linearity
By measuring area responses at different levels of sodium citrate over the range of 25% to 150% of drug
concentration, the linearity data were validated. The solutions were prepared from stock solutions of

STUDY OF CITRATE AND FORMATE 846 T.Kaleemullah et al.

 Vol.4, No.4 (2011), 844-852

required concentration for six different levels of 10.8, 21.6, 32.8, 43.1, 53.4, 64.7µg mL-1. The area and
concentration were treated by least squares linear regression analysis plot [Area counts (AU) at Y-axis Vs
Concentration (µg mL -1) at X-axis] as Figure 3.The calculated values obtained from the 6 calibration
points of linearity data are Slope (0.7749), STEY X (0.2910) and correlation coefficient (0.9999)
respectively.

Fig.-2: Zoomed chromatogram obtained from (a) Sodium citrate standard, (b) PFI sample spiked with its related
substance, (c) NFI sample spiked with its related substance, and (d) Diluent

Precision
The system precision for the method was assessed individually by six replicate injections of sodium
citrate standard solution into chromatographic system, and the percentage relative standard deviation of
response for six replicate measurements was found to be 0.6. Repeatability of the method (Method
precision) was demonstrated by preparing six replicate sample preparations of NFI and PFI dosage form
by without spiking sodium citrate. The samples were analyzed as per method, and the content was
determined for Sodium citrate in individual preparation was found to 3.45%w/w and 3.52%w/w (average
of n=6 determination) for NFI and PFI with RSD values found to be 3.6% and 2.2% respectively.
Intermediate precision of the method (Ruggedness) was performed in the same way as described in
method precision, however, by employing different analyst on other day using another lot of
chromatograph. The content of sodium citrate was determined in each individual preparation separately
for the above pharmaceuticals, and the percentage relative standard deviation for six replicate
measurements in individual preparation was found to was found to 3.52%w/w and 3.42%w/w (average of
n=6 determination) for NFI and PFI with RSD values found to be 4.6% and 4.3% respectively.
Accuracy
The accuracy of the method was evaluated by preparing sample solution spiked with known amount of
sodium citrate at different concentration levels in the range between 80%, 100% and, 120% for NFI and
PFI samples individually. The percent recoveries of sodium citrate were calculated against the known
added amount were summarized in the Table-1.
Stability Studies
To present stability studies of sodium citrate the injectable samples of Nafcillin and Penicillin G
potassium from variable sources of temperature and humidity storage of accelerated (40°C/75%RH), long
term (25°C/60%RH) storage condition28 has been selected. The results obtained from accelerated storage

STUDY OF CITRATE AND FORMATE 847 T.Kaleemullah et al.

 Vol.4, No.4 (2011), 844-852

condition of 6 months samples as well as 24 month long term sample were found to lie within the propose
limit (between 3.4 to 3.8%) for sodium citrate. Hence the sodium citrate resulting as process related
matter, in view of that the sample shows no degradation profile with respect to storage at different
conditions of temperature and humidity.

Fig.-3: Calibration plot obtained for Sodium citrate

Table-1: Accuracy experimental data for Sodium citrate

Components Sodium citrate in NFI Sodium citrate in PFI

Target Level (%) 80 100 120 80 100 120
Spike Conc. (µg/g) a 3.375 4.178 5.164 2.821 3.558 4.296
Determined Conc.(µg/g) a 3.537 4.190 5.498 2.893 3.590 4.415
%RSD a 1.8 3.0 1.0 2.4 1.6 2.1
Percent Recovery a 104.8 100.3 106.5 102.6 100.9 102.7
Average % Recovery 103.8 102.1
95% CI for overall analysis ±3.4 ±1.1
a
Average of n=3 determinations

Validation of Formic acid as Formate

Selectivity
The specificity of the method demonstrated for Ampicillin Trihydrate (APT) drug substance for formic
acid determination. As the sample was found to contain very low level of formic acid in the drug moiety,
so the sample was spiked with formic acid. The retention of the sample and standard were compared. The
retention time of formate was found to be about 3.0 min. Further the sample was spiked with all known
related substance at 0.1%w/w level along with formic acid. The sample shows no co-eluting peaks with
the analyte peak. From the overall experiment, the representative chromatogram obtained from (a) Formic
acid standard, (b) APT drug substance spiked with formic acid along its related substances, and (c)
Diluent shown in Figure 4. The overall %RSD was found to be very much within the acceptance criteria

STUDY OF CITRATE AND FORMATE 848 T.Kaleemullah et al.

 Vol.4, No.4 (2011), 844-852

(Less than 10%) for the investigation samples, thereby indicating that the method is selective for
determining the content of formate in APT drug substance.
Linearity
Different solution of formic acid standard solution prepared over the range of 12% to 120% of drug
concentration at six different concentration levels 1.502, 3.004, 6.008, 9.012, 12.516 & 15.020µg mL-1
from the stock solution. The area and concentration were treated by least squares linear regression
analysis plot [Area counts (AU) at Y-axis Vs Concentration (µg mL -1) at X-axis] as Figure 5.The
calculated values obtained from the calibration points were tabulated for both citric acid and formic acid
in Table-2.

Fig.-4: Zoomed chromatogram obtained from (a) Formic acid standard, (b) APT drug substance spiked with formic
acid along its related substances, and (c) Diluent

Sensitivity
The solutions were prepared from known stock concentration of formic acid to predict the limit of
detection (LOD) and limit of quantification (LOQ).The values were predicted using slope (S) and residual
standard deviation (S.D) obtained from a linear regression line performed at lower concentration levels.
The predicted limit of detection and quantification was calculated using the formula
3.3*STEY.X/SLOPE*100/Sample concentration (for LOD) and 10*STEY.X/Slope*100/Sample
concentration (for LOQ), and each predicted level was verified for precision by analyzing six replicate
measurements. The percentage relative standard deviation for six replicate measurements at predicted
LOD and LOQ concentration levels was found to be within the acceptance limit were summarized in
Table 2.
Precision
The system precision for the method was assessed individually by six replicate injections of Formic acid
(0.12%w/w) standard solution into chromatographic system, and the percentage relative standard
deviation of response for six replicate measurements was found to be 0.3. Repeatability of the method
(Method precision) was demonstrated by preparing six replicate sample preparations of APT drug
substance with formic acid spiking at 0.12%w/w level. The samples were analyzed as per method, and the
content was found to be 0.14%w/w with 3.6% as RSD for n=6 determination. Intermediate precision of
the method (Ruggedness) was performed in the same way as described in method precision, however, by

STUDY OF CITRATE AND FORMATE 849 T.Kaleemullah et al.

 Vol.4, No.4 (2011), 844-852

employing different analyst on other day using another lot of chromatograph. The content of formic acid
was determined in each preparation was found to be 0.14%w/w, and the percentage relative standard
deviation for six replicate measurements determined for each preparation was found to be 3.0%.

Table-2: Evaluation of Linearity for Formic acid

Components Formic acid

Calibration points 6
Slope 40.582
STEY X 0.1960
Correlation Co-efficient 0.9998
RSQ(r2) 0.9996
Limit of detection (µg/g) 0.048
Limit of quantification (µg/g) 0.016
Limit of detection precision (%RSD)b 8.1
Limit of quantification precision(%RSD) b 6.0
b
Average of n=6 determinations

Fig.-5: Calibration plot obtained for Formic acid

Accuracy
The accuracy of the method was evaluated by preparing sample solution spiked with known amount of
formic acid at different concentration levels in the range between LOQ to 150% in APT drug substance.
Each concentration of sample solution was prepared in triplicate and analyzed as per the method. The
percent recoveries of formic acid were calculated against the known added amount and summarized in the
Table-3.
Stability Studies
To present stability studies of formic acid content in Ampicillin trihydrate drug substance for 6 month
accelerated and 36 long term storage sample has collected in random selection. The experimental values
were found to be below limit of quantification (0.06%w/w). Hence formic acid resulting as process
related matter, in view of that the sample shows no degradation profile.

STUDY OF CITRATE AND FORMATE 850 T.Kaleemullah et al.

 Vol.4, No.4 (2011), 844-852

Robustness study for citrate and formate

To assess the robustness of the method with individual determination of citrate and formate, experimental
conditions were deliberately altered. The experimental condition altered by changing the flow of mobile
phase (1.0 mL min-1 ± 10%), temperature (25°C±5°C) and buffer concentration (0.05mM±0.01mM). The
result obtained from the robustness indicated that, the experimental method parameters were tolerance
limit with minor changes to optimize the method.

Table-3: Experimental data obtained from accuracy for Formic acid

Components Formic acid in APT

Target Level (%) 80 100 120
c
Spike Conc. (µg/g) 0.251 0.503 0.750
c
Determined Conc.(µg/g) 0.252 0.504 0.757
c
%RSD 0.15 0.12 0.14
c
Percent Recovery 100.4 100.2 100.9
Average % Recovery 100.5
95% CI for overall analysis ±0.4
c
Average of n=3 determinations

CONCLUSION
A new, accurate and simple ion chromatographic method was proposed for the determination of sodium
citrate in Nafcillin for injection and penicillin G potassium for injection and Formic acid content in
Ampicillin trihydrate drug substance validated as per the ICH guidelines. All statistical results
(Percentage, Mean, RSD, Percentage difference and recovery %) were within the acceptance criteria.

ACKNOWLEDGEMENTS
The authors express their sincere thanks to Islamiah College, Vaniyambadi (Tamil Nadu) and Aurobindo
Pharma limited Research centre, Hyderabad, India for their co-operation.

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