Add the following:

Cosyntropin

(koe'' sin troe' pin).

C136H210N40O31S 2933

1-24-Corticotropin

[16960-16-0].

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 (C136H210N40O31S), calculated on the anhydrous, acetic acid-free basis.

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.

• 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 hydrochloride, 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

Time (min)	Solution A (%)	Solution B (%)
0	100	0
0.5	99	1
18	95	5
19	91	9
29.5	77	23
40	77	23
40.01	40	60
50	40	60

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.

Chromatographic system

(See Chromatography

621

, System Suitability.)

Mode: LC

Detector: UV 254 nm

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

Column temperature: 40

Flow rate: 1 mL/min

Injection volume: 20 µL

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 × 100 ± 90%, where hp is the height of the minor peak, and hv is the height of the valley between the peaks.

]

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

ASSAY

• 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

Chromatographic system

(See Chromatography

621

, System Suitability.)

Mode: LC

Detector: UV 280 nm

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

Column temperature: 40

Flow rate: 1.0 mL/min

Injection volume: 50 µL

Run time: 50 min

System suitability

Sample: Standard solution

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

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of cosyntropin (C136H210N40O31S) in the portion of Cosyntropin taken:

Result = (rU/rS) × (CS/CU) × 100

rU	=	= peak response of cosyntropin from the Sample solution
rS	=	= peak response of cosyntropin from the Standard solution
CS	=	= concentration of cosyntropin in each vial of USP Cosyntropin Acetate RS in the Standard solution (mg/mL)
CU	=	= 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

IMPURITIES

• 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.

System suitability

Samples: Standard solution and Peak identification solution

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

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 = (ri/rT) × 100

ri	=	= peak response of any individual impurity from the Sample solution
rT	=	= sum of all peak responses from the Sample solution

Acceptance criteria

Individual impurities: See Table 2.

Table 2

Name	Relative Retention Time	Acceptance Criteria, NMT (%)
Cosyntropin sulfoxide	0.4	2
Reduced Trp in cosyntropin	0.9	2
Any other individual impurity	—	1
Total impurities	—	5

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.

SPECIFIC TESTS

• UV Absorption Spectrophotometry

(See Spectrophotometry and Light-Scattering

851

, Absorption Spectrophotometry.)

Wavelength range: 240–280 nm

Sample solution: 0.2 mg/mL in 0.1 M hydrochloride

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

: It contains NMT 10 USP Endotoxin Units/mg of cosyntropin.

• Microbial Enumeration Tests

and Tests for Specified Microorganisms

: The total aerobic microbial count is less than 100 cfu/g.

• Water Determination, Method 1c

921

: NMT 10.0%

ADDITIONAL REQUIREMENTS

• Packaging and Storage: Preserve in tight, light-resistant containers at 2

–8

• USP Reference Standards

USP Cosyntropin Acetate RS

USP Endotoxin RS

USP38

1 A suitable substitute is Eluant A from Waters Corporation, catalog number WAT052890.

Auxiliary Information— Please check for your question in the FAQs before contacting USP.

Topic/Question	Contact	Expert Committee
Monograph	Dale R Schmidt Science and Standards Liaison (301) 230-7458	(BIO12010) Monographs - Biologics and Biotechnology 1
85	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison (301) 816-8339	(GCM2010) General Chapters - Microbiology
61	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison (301) 816-8339	(GCM2010) General Chapters - Microbiology
62	Radhakrishna S Tirumalai, Ph.D. Principal Scientific Liaison (301) 816-8339	(GCM2010) General Chapters - Microbiology
Reference Standards	RS Technical Services 1-301-816-8129 rstech@usp.org

USP38–NF33 Page 2958

Pharmacopeial Forum: Volume No. 39(2)