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LABORATORY INFORMATION BULLETIN
DFS/ORO/ORA No. 3978
Drugs
Page 1 of 6
AN HPLC DETERMINATIVE STEP FOR
PRIMIDONE TABLET DISSOLUTION
Andrew G. Agnew
Baltimore District Laboratory
An HPLC method for quantitating Primidone dissolution solutions is described. The method is based on an earlier method1. proposed for the assay of Primidone tablets.
The current USP monograph2 for Primidone Tablets requires a UV determinative step for dissolution, which is subject to interference from excipients. In addition, Primidone has a low specific absorbance at the wavelength of maximum absorbance (A11 = 9.3 at 258 nm, in water). Dilute solutions, such as those produced by a 50 mg tablet in 900 mL of dissolution media (water), are thus more susceptible to error. HPLC separates Primidone from these excipients and concentrates the analyte, yielding more accurate results than direct UV.
Chromatographic System:
Three separate systems were used to test the ruggedness of the method. The general requirements of the system are as follows:
column - C-18, 10 æm particle size, 250 mm long X 4.6 mm diameter, or equivalent
detector - variable wavelength, capable of 258 nm
mobile phase - methanol/water 45/55
flow rate - 1.0 mL/min.
sample injection - 100 uL
NOTE:THE LABORATORY INFORMATION BULLETIN IS A TOOL FOR THE RAPID DISSEMINATION OF LABORATORY METHODS (OR INFORMATION) WHICH APPEAR TO WORK. IT DOES NOT REPORT COMPLETED SCIENTIFIC WORK. THE USER MUST ASSURE HIMSELF BY APPROPRIATE VALIDATION PROCEDURES THAT LIB METHODS AND TECHNIQUES ARE ACCURATE AND RELIABLE FOR HIS INTENDED USE. REFERENCE TO ANY COMMERCIAL MATERIALS, EQUIPMENT, OR PROCESS DOES NOT IN ANY WAY CONSTITUTE APPROVAL, ENDORSEMENT, OR RECOMMENDATION BY THE FOOD AND DRUG ADMINISTRATION.
DFS/ORO/ORA No.3978
Drugs
Page 2 of 6
Equipment:
System 1 - Alltech Econosil C-18 column, 250 mm x 4.6 mm, 10 æm particle size; Spectroflow 757 detector; Perkin Elmer Series 4 HPLC pump; Perkin Elmer ISS 100 auto-injector.
System 2 - Waters æBondapak C-18 column, 300 mm X 3.9 mm, 10 æm particle size; Applied Biosystems 783A detector; Perkin Elmer Series 410 HPLC pump; Perkin Elmer ISS 100 auto-injector.
System 3 - Supelcosil LC-18 column, 250 mm X 4.6 mm, 5 æm particle size; Hewlett-Packard Series 1050 HPLC system (detector, pump, and auto-sampler)
filters - Gelman Supor 450, 0.45 æm membrane.
water - through Milli-Q Plus filter system.
methanol - EM Science OmniSolv, HPLC grade.
Experimental:
Twenty tablets (50 mg Primidone per tablet) were accurately weighed, then composited by grinding and sifting through a 60 mesh sieve. Four separate samples were accurately weighed out, two being about one tablet weight and two being about one half of a tablet weight. The latter two were spiked with USP Primidone to make them equivalent to a whole tablet (approximately 25 mg Primidone being added to each). Each sample was placed in a 900 mL volumetric flask with about 700 mL of HPLC quality water, sonicated for thirty minutes, allowed to cool, filled to the mark with water, shaken well and filtered through a 0.45 æm filter. Duplicate injections were made on each of the three HPLC systems mentioned earlier.
DFS/ORO/ORA No.3978
Drugs
Page 3 of 6
Calculations:
mg Primidone in composite (Q)
= Rspc/Rstd x C x DF
% Recovery of Primidone in spike
= [(Rsps/Rstd x C x DF) - (Sc/W x Q)]/Sp x 100
where:
Rspc = avg. peak response of duplicate composite sample injections
Rsps = avg. peak response of duplicate spike sample
injections
Rstd = avg. peak response of standard injections
C = concentration of standard solution
DF = dilution factor (900 mL)
Sc = weight of composite in spike
W = avg. tablet weight
Sp = weight of primidone added to spike composite (Sc)
Conclusions:
The results are shown on Table 1, along with the results obtained by UV analysis per USP 23. A typical sample chromatogram, produced from system #2, is shown in Figure 1.
As shown by the results in Table 1, analysis of Primidone by HPLC is more accurate and precise than analysis by direct UV at the levels produced by 50 mg tablets. The UV spectra were greatly affected by background interference. The interference was attributed to tablet excipients because the standard solutions produced a linear correlation coefficient of
DFS/ORO/ORA No.3978
Drugs
Page 4 of 6
0.99985. (0.0111 mg/mL, 0.0555 mg/mL, 0.2776 mg/mL, assayed in duplicate) Chromatography was very good on all three systems, with symmetrical peaks (tailing < 1.6), good response, and
resolution from occasional excipient peaks. The ruggedness of the method is also demonstrated by the similarity of results between the three systems, especially systems 2 and 3.
References:
1.Roberts, Stanley E., "High Performance Chromatographic Determination of Primidone in Tablets", LIB #2578.
2."USP 23/NF 18", United States Pharmacopeial Convention, Inc., p. 1290.
Table 1
PRIMIDONE ASSAY AND SPIKE RECOVERY
Table I
PRIMIDONE ASSAY AND SPIKE RECOVERY
Composite Assay System 1 System 2 System 3 USP 23 1 48.7mg 50.6mg 50.1mg 58.2mg 2 48.5mg 50.4mg 49.8mg 47.5mg Avg. 48.6mg 50.5mg 50.Omg 52.8mg
Spike Recovery 1 * 94.5 % 100.4 % 99.9 % 79.9 % 2** 98.4 % 100.9 % 100.2 % 111.5 % Avg. 96.4 % 100.6 % 100.0 % 95.7 %
*of 25.1 71 mg added. **of 25.387 mg added.
