ASSAY FOR CITRIC ACID/CITRATE AND PHOSPHATE

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 INTRODUCTION

The following ion chromatographic general procedure is provided for the determination of citric acid/citrate and phosphate in compendial articles, when specified in the individual monographs. See lon Chromatography (1065), for discussion of the theory and principles of measurements using ion chromatography.

Change to read:

2 ASSAY

^▲[NOTE-Use procedure A if the monograph does not specify a procedure._{▲(USP 1-MAY-2023)}

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2.1 PROCEDURE ▲A▲(USP 1-MAY-2023)

Mobile phase: 20 mM sodium hydroxide or potassium hydroxide from an appropriate volume of carbonate-free sodium hydroxide or potassium hydroxide solution of known concentration and water (resistivity NLT 18 megohm-cm). Alternatively, Mobile phase can be generated electrolytically using an automatic eluent generator. Protect the Mobile phase from atmospheric carbon dioxide.

Standard solution 1 (for the assay of citric acid/citrate only): 20 µg/mL of citrate (C₆H₅O₇) in freshly prepared 1 mM sodium hydroxide from USP Citric Acid RS

Standard solution 2 (for the concomitant assay of citrate and phosphate): 20 µg/mL of citrate (C₆H₅O₇) and 12 µg/mL of phosphate (PO₄) in freshly prepared 1 mM sodium hydroxide from USP Citric Acid RS and monobasic sodium phosphate

Sample solution (for the assay of citric acid/citrate): Nominally 20 µg/mL of citrate in freshly prepared 1 mM sodium hydroxide, unless otherwise stated in the monograph

Sample solution (for the assay of phosphate): Nominally 12 µg/mL of phosphate in freshly prepared 1 1 mM sodium hydroxide, unless otherwise stated in the monograph

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: Conductivity with suppression

Columns

Analytical: 4-mm x 25-cm; 13-µm packing L61

Guard: 4-mm x 5-cm; 13-µm packing L61

Temperatures

Column: 30°

Detector: 35°

Suppressor: 4-mm membrane anionic autosuppressor or a suitable chemical suppression system

Flow rate: 2 mL/min

Injection volume: 10 µL

System suitability

Samples: Standard solution 1 and/or Standard solution 2, as appropriate [NOTE-The relative retention times for phosphate and citrate are 0.57 and 1.0, respectively.]

Suitability requirements

Tailing factor: NMT 2.0 for the citrate and/or phosphate peaks, as appropriate

Relative standard deviation: NMT 1.5% for six replicate injections for the citrate and/or phosphate peaks, as appropriate

Analysis

Samples: Standard solution 7 and/or Standard solution 2, and Sample solution Unless otherwise stated in the monograph, calculate the concentration of citrate or phosphate in the portion of the Sample solution taken:

                      Result = (r_U/r_S) x C_S

r_U = peak response of citrate or phosphate from the Sample solution

r_S = peak response of citrate or phosphate from Standard solution 1 or Standard solution 2

C_S = concentration of citrate or phosphate from Standard solution 1 or Standard solution 2 (µg/mL)

Add the following:

2.2 Procedure B

Mobile phase, Standard solution 1, Standard solution 2, and Sample solution(s): Prepare as directed in Procedure A.

Chromatographic system

(See Chromatography (621) System Suitability.)

Mode, Detector, Temperatures, Suppressor, and Injection volume: Proceed as directed in Procedure A.

Columns

Guard: 4-mm x 5-cm; 10-um packing L21

Analytical: 4.6-mm x 25-cm; 9-µm packing L46

Flow rate: 1 mL/min

Run time: 15 min

System suitability

Samples: Standard solution 1 and/or Standard solution 2, as appropriate

[NOTE-The relative retention times for phosphate and citrate are 0.65 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 6.5 between phosphate and citrate, Standard solution 2

Tailing factor: NMT 1.5 for citrate and/or phosphate, as appropriate

Relative standard deviation: NMT 1.5% for 6 replicate injections for citrate and/or phosphate, as appropriate

Analysis: Proceed as directed in Procedure A._{▲(USP 1-May-2023)}

3 ADDITIONAL REQUIREMENTS

USP REFERENCE STANDARDS (11)

USP Citric Acid RS