Cosyntropin

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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C₁₃₆H₂₁₀N₄₀O₃₁S                 2933

a^1-24-Corticotropin CAS RN^®: 16960-16-0; UNII: 72YY86EA29.

DEFINITION

Cosyntropin is a synthetic peptide whose sequence is identical to the first 24 amino acids of human adrenocorticotropic hormone (ACTH). Cosyntropin contains NLT 90% and NMT 102% of cosyntropin (C136H210N 40031S), calculated on the anhydrous, acetic acid-free basis.

1 IDENTIFICATION

A. The retention time of the cosyntropin peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

1.1 B. AMINO ACID ANALYSIS

Hydrolysis solution: 12 N hydrochloric acid containing a small crystal (0.1%-1.0%) of phenol

Sample hydrolysate preparation: Transfer a portion of cosyntropin into a vial, weigh it, and dissolve in water to a concentration of 3 mg/mL. Transfer 70 mg of this solution to a vacuum hydrolysis tube. Add 70 µL of Hydrolysis solution, seal the tube, and heat for 24 h at 110°. Cool the tube, and remove the solvents at reduced pressure. Resuspend the hydrolysate residue in 1 mL of 0.020 M hydrochloric acid, and filter.

Solution A: Prepare a solution having a composition of 140 mM sodium acetate and 17 mM triethylamine, and adjust with phosphoric acid to a pH of 5.02.1

Solution B: Acetonitrile and water (60:40)

Mobile phase: See Table 1.

Table 1

Sample solution: Add 140 µL of 0.2 M borate buffer (pH 8.8) to 20 µL of Sample hydrolysate preparation. Add 40 µL of 10 mM 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Incubate for 2 min at room temperature. Transfer to an insert in an HPLC vial, seal with a silicone cap, and incubate for 10 min at 55°.

Standard solution: Prepare a solution having known equimolar amounts of L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tyrosine, and L-valine with half the equimolar amount of L-Cystine. [NOTE-Suitable concentrations are 0.1 mM and 0.05 mM, respectively.] Derivatize with AQC as outlined for Sample solution.

1.1.1 Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 3.9-mm x 15-cm; 4-µm packing L1

Column temperature: 40°

Flow rate: 1 mL/min

Injection volume: 20 µL

1.1.2 System suitability

Samples: Sample solution and Standard solution

Suitability requirements: All 17 Amino acid peaks must be visible in the Standard solution.

Resolution: NLT 1.5 between each of the 17 amino acid peaks in the Standard solution. [NOTE-In those cases where the peak does not recover to the baseline, the following criteria will be applied: (hp-hv)/hp x 100 ± 90%, where hp is the height of the minor peak, and hv is the height of the valley between the peaks.]

1.1.3 Analysis

Samples: Sample solution and Standard solution

First record and measure the responses for each amino acid peak in the Standard solution. Express the content of each amino acid in moles.

Calculate the mean nmol of the amino acids:

                 Result = (nmol found in the Analysis for Glu, Gly, Val, Phe, Lys, Arg, Pro)/17

Divide the nmol of each amino acid by the Result to determine the amino acid ratios that must meet the Acceptance criteria.

Acceptance criteria: 3.5-4.7 of lysine; 0.9-1.1 of histidine; 2.7-3.3 of arginine; 1.1-2.2 of serine; 0.9-1.1 of glutamic acid; 2.5-3.5 of proline; 1.8-2.2 of glycine; 0.9-1.1 of methionine; 1.7-2.2 of tyrosine; 0.9-1.1 of phenylalanine; 2.7-3.3 of valine

2 ASSAY

2.1 PROCEDURE

Mobile phase: 365 mL of acetonitrile, 10 mL of glacial acetic acid, and 10 g of ammonium sulfate. Dilute with water to 2 L, and a pH of about 3.3.

Standard solution: 1.0 mg/mL of USP Cosyntropin Acetate RS

Sample solution: 1.0 mg/mL of Cosyntropin

2.1.1 Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 4.6-mm x 25-cm; 5-µm packing L1

Column temperature: 40°

Flow rate: 1.0 mL/min

Injection volume: 50 µL

Run time: 50 min

2.1.2 System suitability

Sample: Standard solution

2.1.3 Suitability requirements

Resolution: NLT 1.0 between the main cosyntropin peak and the reduced Trp-cosyntropin peak in the Standard solution

Retention time: 16-20 min for the cosyntropin peak, Standard solution

Relative standard deviation: NMT 2.0% for the cosyntropin peak from three replicate injections of the Standard solution

2.1.4 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of cosyntropin (C136H210N 4003,S) in the portion of Cosyntropin taken: 31

                 Result = (r_U/r_S) x (C_S/C_U) × 100

r_U = peak response of cosyntropin from the Sample solution

r_S = peak response of cosyntropin from the Standard solution

C_S = concentration of cosyntropin in each vial of USP Cosyntropin Acetate RS in the Standard solution (mg/mL) C

C_U = concentration of Cosyntropin, calculated on the anhydrous, acetic acid-free basis, in the Sample solution (mg/mL)

Acceptance criteria: 90.0%-102.0% of cosyntropin on the anhydrous, acetic acid-free basis

3 IMPURITIES

3.1 ORGANIC IMPURITIES, RELATED PEPTIDES

Mobile phase, Standard solution, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

Peak identification solution: 1.0 mg/mL of cosyntropin in 1% (v/v) glacial acetic acid. Add 50 µL of 30% hydrogen peroxide:water (1:999). Let stand for 2 h to produce the main impurity, cosyntropin sulfoxide.

3.1.1 System suitability

Samples: Standard solution and Peak identification solution

3.1.2 Suitability requirements

Resolution: NLT 1.0 between the main cosyntropin peak and the reduced Trp-cosyntropin peak in the Standard solution

Retention time: 16-20 min for the cosyntropin peak, Standard solution

Relative retention time of main impurity in Peak identification solution: About 0.4

Relative standard deviation: NMT 2.0% for the cosyntropin peak from three replicate injections of the Standard solution

3.1.3 Analysis

Sample: Sample solution

Integrate the chromatogram using the normalization procedure.

Calculate the percentage of cosyntropin-related impurities in the portion of Cosyntropin taken:

                 Result = (r_i/r_T) × 100

r_i = peak response of any individual impurity from the Sample solution

r_T = sum of all peak responses from the Sample solution

3.1.4 Acceptance criteria

Individual impurities: See Table 2.

Table 2

4 OTHER COMPONENTS

ACETIC ACID IN PEPTIDES (503): 8%-13% using a 1-mg/mL Test Solution prepared by dissolving 10 mg of Cosyntropin in 10 mL of Solution A and

Solution B (95:5)

TRIFLUOROACETIC ACID CONTENT: Perform the method contained in (503), substituting a suitable quantity of trifluoroacetic acid for the Standard solution. The portion of Cosyntropin taken shall contain NMT 0.5% trifluoroacetic acid.

5 SPECIFIC TESTS

UV ABSORPTION SPECTROPHOTOMETRY

(See Ultraviolet-Visible Spectroscopy (857).)

Wavelength range: 240-280 nm

Sample solution: 0.2 mg/mL in 0.1 M hydrochloric acid

Acceptance criteria: Absorptivity of 0.51-0.61, calculated on the anhydrous and acetic acid-free basis, at the maximum wavelength of 276 nm

Ratio: A276/A248, 2.4-2.9

BACTERIAL ENDOTOXINS TEST (85): It contains NMT 10 USP Endotoxin Units/mg of cosyntropin.

MICROBIAL ENUMERATION TESTS (61) and TESTS FOR SPECIFIED MICROORGANISMS (62): The total aerobic microbial count is less than 100 cfu/g.

WATER DETERMINATION, Method 1c(921): NMT 10.0%

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight, light-resistant containers at 2"-8".

USP REFERENCE STANDARDS (11)

USP Cosyntropin Acetate RS