test 1—
Mobile phase and Chromatographic system—
Prepare as directed in the Assay.
Standard piperacillin solution—
Transfer about 40 mg of
USP Piperacillin RS, accurately weighed, to a 100-mL volumetric flask, add a few drops of methanol to dissolve it, dilute with
Mobile phase to volume, and mix. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, dilute with
Mobile phase to volume, and mix.
[note—Use these solutions within 1 hour.
]
Standard ampicillin solution—
Prepare a solution of
USP Ampicillin RS in
Mobile phase having a known concentration of about 0.08 mg per mL.
Test solution—
Use the Assay preparation.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Test solution, the
Standard ampicillin solution, and the
Standard piperacillin solution, and proceed as directed in the
Assay. Calculate the percentage of ampicillin in the portion of Piperacillin taken by the formula:
10C(P / W)(rU / rSa)
in which
C is the concentration, in mg per mL, of
USP Ampicillin RS in the
Standard ampicillin solution; P is the designated potency, in µg of ampicillin per mg, of
USP Ampicillin RS;
W is the weight, in mg, of Piperacillin taken to prepare the
Test solution; and
rU and
rSa are the peak responses of ampicillin obtained from the
Test solution and the
Standard ampicillin solution, respectively: not more than 0.2% of ampicillin is found.
Calculate the percentage of piperacillin related compound A (4-carboxy-
-[2-(4-ethyl-2,3-dioxo-1-piperazinecarboxamido-2-phenylacetamido]-5,5-dimethyl-2-thiazolidinacetic acid), piperacillin related compound B (1-ethyl-2,3-piperazinedione), and piperacillin related compound C (2-(3-acetyl-4-carboxy-5,5-dimethyl-2-thiazolidinyl)-N-[N-[(4-ethyl-2,3-dioxo-1-piperazinyl)-carbonyl]-2-phenylglycyl]glycine) in the portion of Piperacillin taken by the formula:
10C(P / W)(RRFi)(ri / rSp)
in which
C is the concentration, in mg per mL, of
USP Piperacillin RS in the
Standard piperacillin solution; P is the designated potency, in µg of piperacillin per mg, of
USP Piperacillin RS;
W is the weight, in mg, of Piperacillin taken to prepare the
Test solution; RRFi is the response factor of an individual piperacillin related compound relative to the response of piperacillin, specifically 1.4 for piperacillin related compound A, 0.41 for piperacillin related compound B, and 0.93 for piperacillin related compound C;
ri is the response of each impurity peak obtained from the
Test solution; and
rSp is the peak response of piperacillin obtained from the
Standard piperacillin solution: not more than 1.0% of piperacillin related compound A, not more than 0.2% of piperacillin related compound B, and not more than 0.4% of piperacillin related compound C is found.
test 2—
Mobile phase—
Prepare a mixture of methanol, water, 0.2 M monobasic sodium phosphate, and 0.4 M tetrabutylammonium hydroxide (615:282:100:3). Adjust with phosphoric acid to a pH of 5.50 ± 0.02, and degas. Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Standard stock solution—
Transfer about 20 mg of
USP Piperacillin RS, accurately weighed, to a 50-mL volumetric flask, add a few drops of methanol to dissolve it, dilute with
Mobile phase to volume, and mix.
[note—Use this solution within 1 hour.
]
Standard solution—
Transfer 5.0 mL of the Standard stock solution to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix. [note—Use this solution within 1 hour.]
Test solution—
Transfer about 40 mg of Piperacillin, accurately weighed, to a 100-mL volumetric flask, add a few drops of methanol to dissolve it, dilute with Mobile phase to volume, and mix. [note—Use this solution within 1 hour.]
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the relative retention times are about 1.0 for piperacillin and 2.55 for 6-[2-[6-[2-(4-ethyl-2,3-dioxo-1-piperazinecarboxamido)-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxamido]-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (piperacillin related compound D); and the relative standard deviation for replicate injections is not more than 2%.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard solution and the
Test solution into the chromatograph, record the chromatograms, and measure the peak responses for piperacillin and piperacillin related compound D. Calculate the percentage of piperacillin related compound D in the portion of Piperacillin taken by the formula:
10C(1.47)(P / W)(ri / rS)
in which
C is the concentration, in mg per mL, of
USP Piperacillin RS in the
Standard solution; 1.47 is the relative response factor for piperacillin related compound D;
P is the designated potency, in µg of piperacillin per mg, of
USP Piperacillin RS;
W is the weight, in mg, of Piperacillin taken to prepare the
Test solution; ri is the impurity peak response obtained from the
Test solution; and
rS is the peak response of piperacillin obtained from the
Standard solution: not more than 2.0% of piperacillin related compound D is found. The sum of all impurities found in
Test 1 and
Test 2 is not more than 3.8%.
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