Application Note 257

HPLC Assay Method for Drug Products

Containing Anti-Tuberculosis Active
Pharmaceutical Ingredients
Introduction compounds and the other drug product contains three
Isoniazid, pyrazinamide, rifampicin, and ethambutol of the compounds (no ethambutol). The 10 min HPLC
are anti-tuberculosis compounds. The standard method accurately determines all compounds of interest
treatment for tuberculosis (TB) is to treat the patient in both drug products. This method saves time, reduces
with a combination of these four compounds for two mobile phase consumption, and reduces waste. Further
months, followed by isoniazid and rifampicin alone for savings and waste reduction are possible with an ultra
an additional four months. Depending on the state of HPLC (UHPLC) method that requires less than 2 min
infection, ethambutol may be omitted from the treatment.1 per injection.
These compounds are used in combination because they
EQUIPMENT
have different modes of action. For more than 50 years,
Dionex UltiMate® 3000 system including:
TB has been treated with combination drug therapy and
there are a number of available combination drug products Equipment Conventional UHPLC
with different drug contents and composition. HPLC
The United States Pharmacopeia (USP) 32 NF27 Integrated vacuum degasser SRD-3600 SRD-3600
solvent rack
contains a monograph for isoniazid, pyrazinamide,
Pump DGP-3600A HPG-3400RS
rifampicin, and ethambutol hydrochloride tablets.2
The monograph has two assay methods for this drug Split-loop sampler WPS-3000TSL WPS-3000TRS

product. One method is for an assay of isoniazid, Column compartment TCC-3200 TCC-3000RS

pyrazinamide, and rifampicin and the other for Diode array detector PDA-3000 DAD-3000RS
ethambutol. Both are HPLC methods. Sample loop size 100 µL 100 µL*
The work shown here reports a single HPLC assay Mixer Standard 200 µL Static mixer kit
method that accurately determines these four compounds. Flow cell 13 µL SST 2.5 µL SST
The method is evaluated using two drug products. Chromeleon® Chromatography 6.80 SR7 6.80 SR7
One drug product is a tablet containing all four Data System (CDS) software
version

*While the data collected in this AN used a 100 µL loop, a smaller loop
(e.g. 20 µL) would be more appropriate.
REAGENTS AND STANDARDS UHPLC
Deionized water (DI), Type I reagent grade, 18 MΩ-cm Column: Acclaim PA2, 2.2 µm 2.1 × 100 mm
resistivity or better (P/N 068990)
Acetonitrile (CH3CN), HPLC grade (LAB-SCAN) EXP™ Pre-Column Ultra High
Sodium dihydrogen orthophosphate, AR grade (Ajax) Pressure Filter Cartridges, 0.2µm
Triethylamine (TEA), AR grade (Fisher) (P/N 15-04-03097, Optimize
Orthophosphoric acid, (85%), AR grade (ASP Finechem) Technologies)
Isoniazid, (101.30%), (provided by customer) EXP Filter Holder with EXP™
Titanium Hybrid Ferrule
Pyrazinamide, (99.91%), (provided by customer)
(P/N 15-04-03837, Optimize
Ethambutol, (98.10%), (provided by customer) Technologies)
Rifampicin, (96.92%), (provided by customer) Mobile Phase: A: 4% CH3CN in 20
CONDITIONS mM NaH2PO4 (plus 1.5 mL TEA per
liter), pH 6.8
Conventional HPLC B: 50% CH3CN in 20 mM NaH2PO4
Column: Acclaim® Polar Advantage II (PA2), (plus 1.5 mL TEA per liter), pH 6.8
3 µm 4.6 × 150 mm (P/N 063191) Flow Rate: 1.0 mL/min
Acclaim PA2 Guard, 5 µm Gradient: 100% A from -2.5 to 0.5 min, ramp to
4.3 × 10 mm (P/N 063195) 100% B in 0.1 min, and hold 100% B
Acclaim Guard Kit (P/N 059526) for 1.2 min
Mobile Phase: A: 8% CH3CN in 20 mM NaH2PO4 Column Temp.: 35 °C
(plus 1.5 mL TEA per liter), pH 6.8 Injection Volume: 1.5 µL
B: 50% CH3CN in 20 mM NaH2PO4 Detection: Channel_1 UV-vis_1 at 200 nm and
(plus 1.5 mL TEA per liter), pH 6.8 337 nm at 1 min
Flow Rate: 1.0 mL/min Channel_2 UV-vis_2 at 238 nm
Gradient: 100% A from -5 to 3 min, ramp to Wavelength scanning 190 to 800 nm
100% B in 0.5 min, and hold 100% B Data collection rate 25 Hz, response
for 7 min time 0.2 s
Column Temp.: 35 °C
PREPARATION OF SOLUTIONS AND REAGENTS
Injection Volume: 5 µL
Detection: Channel_1 UV-vis_1 at 200 nm and 20 mM NaH2PO4 pH 6.8 plus 1.5 mL TEA
337 nm at 5 min Dissolve 3.12 g NaH2PO4 in 700 mL water, add
Channel_2 UV-vis_2 at 238 nm 1.5 mL TEA, and mix well. Transfer this solution into a
Wavelength scanning 190 to 800 nm 1 L volumetric flask and add water to bring to volume.
Data collection rate 5 Hz, rise time 0.5 s Adjust to pH 6.8 with orthophosphoric acid.
Mobile Phases
Mobile Phase A (Conventional HPLC)
Mix 80 mL CH3CN with 920 mL of 20 mM NaH2PO4
TEA pH 6.8. Filter with a 0.2 µM filter.
Mobile Phase A (UHPLC)
Mix 40 mL CH3CN with 960 mL of of 20 mM
NaH2PO4 TEA pH 6.8. Filter with a 0.2 μM filter.
Mobile Phase B
Mix 500 mL CH3CN with 500 mL of 20 mM
NaH2PO4 TEA pH 6.8. Filter with a 0.2 µM filter.

2 HPLC Assay Method for Drug Products Containing Anti-Tuberculosis Active Pharmaceutical Ingredients
Stock Standard Solutions pyrazinamide, ethambutol, and rifampicin. Sample B
Accurately weigh 20 mg, 110 mg, 74 mg, and 110 mg had only three of the APIs; it lacked ethambutol. Table 2
isoniazid, pyazinamide, ethambutol, and rifampicin, reports the content of each drug and API concentration
respectively, into a 50 mL beaker. Add 5 mL CH3CN and after sample preparation.
20 mL mobile phase A. Stir and place in an ultrasonic
Sample A
bath until dissolution is complete. Transfer this solution
1. Grind a tablet of Sample A and transfer into a 50 mL
to a 50 mL volumetric flask, rinse the beaker with mobile
beaker. Add 5 mL CH3CN and 20 mL mobile phase
phase A a few times, and transfer into the same volumetric
A. Stir and place in an ultrasonic bath until dissolution
flask. Add mobile phase A to bring to volume. Table 1
is complete. Transfer this solution into a 100 mL
shows the concentration of the stock standard solution.
volumetric flask, rinse the beaker with mobile phase A
Working Standard Solutions a few times, and transfer into the same volumetric
Pipet 1, 1.5, and 2 mL stock standard solution into flask. Add mobile phase A to bring to volume.
separate 10 mL volumetric flasks. Add mobile phase A to 2. Pipet 0.75 mL of this sample solution into a 10 mL
bring to volume. Table 1 shows the concentrations of the volumetric flask and add mobile phase A to bring to
working standard solutions. volume. Filter with a 0.2 µm filter.
Note: Prepare stock and working standard solutions
Sample B
just before analysis.
1. Prepare a tablet in the same manner as step 1
Sample Preparation for Sample A.
The authors analyzed two different anti-tuberculosis 2. Pipet 1 mL of this sample solution into a 10 mL
drug samples (referred to as Samples A and B) and these volumetric flask and add mobile phase B to bring to
drugs had different compositions. Sample A contained volume. Filter with a 0.2 µm filter.
four active pharmaceutical ingredients (API): isoniazid, Note: Prepare samples on the day of analysis.

Table 1. Concentrations of Stock and Working Standard Solutions

Concentration of Working Standard Solution
Stock Standard Solution Volume (mL)*
Compound Stock Standard Concentration (mg/L)
Solution (mg/L) L1 L2 L3 L1 L2 L3
Isoniazid 405 1 1.5 2 40.5 60.8 81.0
Pyrazinamide 2198 1 1.5 2 220 330 440
Ethambutol 1452 1 1.5 2 145 218 290
Rifampicin 775 1 1.5 2 78 116 155

*Volume used to prepare 10 mL of working standard solution

Table 2. Tablet Content and Sample Concentration after Sample Preparation

Sample A Sample B
Compound Calculated Concentration Calculated Concentration
Tablet Content
Tablet Content (mg/tablet) after Sample Preparation after Sample Preparation
(mg/tablet)
(mg/L) (mg/L)
Isoniazid 75 56.3 80 80
Pyrazinamide 400 300 250 250
Ethambutol 275 206 — —
Rifampicin 150 113 120 120

Application Note 257 3

 Table 3. Tablet Weights 2,500 UV_vis_1
Column: Acclaim PA2 3 µm, 4.6 × 150 mm
UV_vis_2 Eluent: A: 8% CH3CN in 20 mM NaH2PO4
Tablet Weight (g) (plus 1.5 mL of TEA), pH 6.8
Tablet No. B: 50% CH3CN in 20 mM NaH2PO4
Sample A Sample B (plus 1.5 mL of TEA), pH 6.8
1 1.23 0.71 2 Flow Rate: 1.0 mL/min
Gradient: 100% A from -5 to 3 min, ramp to 100% B
2 1.19 0.72 in 0.5 min and hold 100% B for 7 min
Temperature: 35 °C
3 1.20 0.70 Inj. Volume: 5 µL
Detection: UV_vis_1 at 200 nm and 337 nm at 5 min
Average 1.21 0.71 UV_vis_2 at 238 nm
Sample: Mixture of four anti-tuberculosis
Total Weight of APIs (g) 0.91 0.45 drugs standard
Placebo Weight (g) 0.30 0.26
mAU Peaks: Conc. (mg/L)
1. Isoniazid 81
2. Pyrazinamide 440
3. Ethambutol 290
Table 4. Spiked Sample Concentrations 4. Rifampicin 155

Calculated Spiked
Spiked Standard Concentration after
Amount (mg) Sample Preparation 4

Compound (mg/L) 1
In 0.3 g of In 0.26 g Spiked Spiked
Sample A of Sample Sample A Sample B
Placebo B Placebo Placebo Placebo 3
Isoniazid 75 80 57 81
Pyrazinamide 400 250 300 250 0 1.3 2.5 3.8 5 6.3 7.5 8.8 10.5
Minutes 27670
Ethambutol 275 — 202 —
Rifampicin 150 120 109 116 Figure 1. Chromatograms of a standard containing four
anti-tuberculosis drugs.

Spiked Placebo Sample hydrochloride tablets. The USP monograph has two assay
To calculate the placebo weight for each sample, methods. The first assay is for isoniazid, pyrazinamide,
average the weights of the three tablets and subtract the and rifampicin. It is an HPLC assay that calls for a
average total API weight of those tablets to obtain the 4.6 × 250 mm, 5 µm L1 column, a sodium phosphate
average placebo weight (Table 3). Use the same placebo buffer pH 6.8/CH3CN eluent, and a 238 nm detection
weight for each sample in Table 3 for the spiked placebo wavelength. The second assay, which is also an HPLC assay,
sample preparation. Spike standards (dry) into the is for ethambutol. This assay calls for a 4.6 × 150 mm,
placebo to achieve API content similar to the tablet 5 µm L10 column, triethylamine pH 7/CH3CN eluent,
content. Table 4 shows the amount of standards added and a 200 nm detection wavelength. To create a single
to each sample placebo and the calculated concentration method for all four APIs, the authors used an Acclaim PA2
after sample preparation. (3 µm, 4.6 × 150 mm) column with a sodium phosphate
plus triethylamine pH 6.8/CH3CN eluent. Figure 1
RESULTS AND DISCUSSION
shows a separation of all four compounds in 10 min.
Separation and Detection The compounds were detected with two UV detection
The goal of this work was to create one method to channels. Channel 1 (UV-vis_1) detects compounds by
determine all four APIs in the combination drug product. absorbance at 200 nm for the first 5 min and at 337 nm
The authors started by reviewing the USP monograph for the final five min. Channel 2 (UV-vis_2) detects at
for rifampin, isoniazid, pyrazinamide, and ethambutol 238 nm. Ethambutol is not detected at 238 nm.

4 HPLC Assay Method for Drug Products Containing Anti-Tuberculosis Active Pharmaceutical Ingredients
 Table 5. Calibration Results at UV-vis_1 as Reported 1,600 UV_vis_1 Column: Acclaim PA2 3 µm, 4.6 × 150 mm
by Chromeleon Software UV_vis_2
2
Eluent: A: 8% CH3CN in 20 mM NaH2PO4
(plus 1.5 mL of TEA), pH 6.8
Cal. B: 50% CH3CN in 20 mM NaH2PO4
Compound Points r2 Offset Slope
Type (plus 1.5 mL of TEA), pH 6.8
Flow Rate: 1.0 mL/min
Isoniazid LOff 3 0.99999 -0.3938 0.2920 Gradient: 100% A from -5 to 3 min, ramp to 100% B
in 0.5 min and hold 100% B for 7 min
Pyrazinamide LOff 3 0.99979 3.1730 0.2684 Temperature: 35 °C
Inj. Volume: 5 µL
Ethambutol LOff 3 0.99938 -0.1861 0.0088 Detection: UV_vis_1 at 200 nm and 337 nm at 5 min
Rifampicin LOff 3 0.99999 -0.4919 0.1338 UV_vis_2 at 238 nm
Sample: Sample A

Peaks: Conc. (mg/L)

mAU
1. Isoniazid 54.8/54.8
2. Pyrazinamide 305/301
Table 6. Calibration Results at UV-vis_2 as Reported 3. Ethambutol 197/–
by Chromeleon Software 4. Rifampicin 120/120

Cal. 4
Compound Points r2 Offset Slope
Type
1
Isoniazid LOff 3 0.99994 -0.1826 0.1153
Pyrazinamide LOff 3 0.99998 -0.4231 0.0812
Ethambutol — — — — — 3

Rifampicin LOff 3 0.99998 -0.7014 0.1801

0 1.3 2.5 3.8 5 6.3 7.5 8.8 10.5

Minutes 27671
Method Calibration
After optimizing sample preparation to determine that Figure 2. Example chromatograms of Sample A.
all compounds can be detected in each of the two samples,
three-point calibration curves were prepared using the two
UV channels with the diode array detector. The calibration Column: Acclaim PA2 3 µm, 4.6 × 150 mm
1,400 Eluent: A: 8% CH3CN in 20 mM NaH2PO4
data in Table 5 show linear peak area response for each UV_vis_1
UV_vis_2
(plus 1.5 mL of TEA), pH 6.8
B: 50% CH3CN in 20 mM NaH2PO4
detected compound in channel 1 and Table 6 shows (plus 1.5 mL of TEA), pH 6.8
2
linear peak area response for each detected compound in Flow Rate: 1.0 mL/min
Gradient: 100% A from -5 to 3 min, ramp to 100% B
channel 2. in 0.5 min and hold 100% B for 7 min
Temperature: 35 °C
Sample Analysis Inj. Volume: 5 µL
Detection: UV_vis_1 at 200 nm and 337 nm at 5 min
Customers provided Samples A and B as well as UV_vis_2 at 238 nm
Sample: Sample B
products without the APIs, referred to as Sample A
Placebo and Sample B Placebo. Three tablets for each Peaks: Conc. (mg/L)
mAU 1. Isoniazid 76.1/76.2
sample were prepared and three injections of each 2. Pyrazinamide 249/248
3. Rifampicin 119/118
prepared tablet were made to evaluate the reproducibility
of sample preparation, injection, and tablet content.
Chromatograms for Samples A and B are shown in 3
1
Figures 2 and 3, respectively. Sample A contained the
expected four APIs, whereas Sample B contained the
expected three APIs. Neither tablet contained compounds
that interfere with determination of the four APIs. To
determine if the four peaks were pure, the photodiode
array detector was used for the standard separation. The
authors injected single component standards, collected the 0 1.3 2.5 3.8 5 6.3 7.5 8.8 10.5
Minutes 27672
spectral data, and entered it into the spectral library.
Figure 3. Example chromatograms of Sample B.

Application Note 257 5

 Table 7. Peak Purity, Resolution, and Peak Analysis Results for the Standard
Resolution* Peak Purity RSD Peak Peak Purity RSD Peak Plates
Compound Asymmetry
(USP) Match Purity Match Index Purity Index (USP)
Isoniazid 4.98 1000 0.47 215.9 0.22 1.42 12410
Pyrazinamide 3.58 1000 0.51 226.2 0.23 1.14 16446
Ethambutol 29.92 995 1.78 194.3 0.80 1.94 7244
Rifampicin n.a. 998 3.45 284.3 1.21 1.20 —

Table 8. Peak Purity, Resolution, and Peak Analysis Results for the Samples
Sample Compound Peak Purity RSD Peak Peak Purity RSD Peak Resolution* Asymmetry Plates Match with
Match Purity Index Purity (USP) (USP) Library
Match Index
Isoniazid 1000 0.49 215.9 0.23 4.96 1.38 12185 1000
Pyrazinamide 1000 0.58 226.2 0.26 3.76 1.16 17480 1000
Sample A
Ethambutol 993 2.57 195.0 1.20 30.46 1.81 7705 999
Rifampicin 997 4.32 284.7 1.50 n.a. 1.16 — 997
Isoniazid 1000 0.37 215.9 0.17 4.94 1.37 11999 1000
Pyrazinamide 1000 0.50 226.3 0.22 47.02 1.14 17053 1000
Sample B
Ethambutol — — — — — — — —
Rifampicin 998 2.25 286.0 0.77 n.a. 1.20 — 996
*Calculation is based on USP and is compared to the next main peak.

The data in Table 7 suggest that each peak in the standard

was pure. Table 8 shows that all peaks in Samples A and
B were pure (judging by the values calculated from the
spectral data) and all peaks had very good spectral match
with the data entered into library, also suggesting that the
peaks in the samples were pure.

6 HPLC Assay Method for Drug Products Containing Anti-Tuberculosis Active Pharmaceutical Ingredients
 Table 9. Average Found Concentration from Three Injections for Three Tablets at UV-vis_1
Sample Compound Calculated Tablet 1 Tablet 2 Tablet 3
Concentration
(mg/L) Average RSD % Content Average RSD % Content Average RSD % Content
Isonaizid 56.3 54.8 0.47 97.34 53.3 0.13 97.26 51.3 0.42 91.12
Pyrazinamide 300 305 0.69 101.7 303 0.09 101.0 288 0.25 96.00
A
Ethambutol 206 197 0.77 95.63 198 0.65 96.12 201 0.65 97.57
Rifampicin 113 120 0.50 106.2 115 0.28 101.8 120 0.21 106.2
Isonaizid 80 76.1 0.08 95.13 76.2 0.16 95.25 73.2 0.42 91.50
Pyrazinamide 250 249 0.23 99.60 251 0.09 100.4 246 0.37 98.4
B
Ethambutol — — — — — — — — — —
Rifampicin 120 119 0.15 99.17 125 0.11 104.2 115 0.14 95.83

Table 10. Average Found Concentration from Three Injections for Three Tablets at UV-vis_2
Sample Compound Calculated Tablet 1 Tablet 2 Tablet 3
Concentration
(mg/L) Average RSD % Content Average RSD % Content Average RSD % Content
Isonaizid 56.3 54.8 0.51 97.34 53.2 0.13 94.49 51.4 0.24 91.30
Pyrazinamide 300 301 0.53 100.3 298 0.10 99.33 284 0.27 94.67
A
Ethambutol 206 — — — — — — — — —
Rifampicin 113 120 0.52 106.2 115 0.28 101.8 120 0.18 106.2
Isonaizid 80 76.2 0.08 95.25 76.3 0.13 95.38 73.2 0.09 91.5
Pyrazinamide 250 248 0.15 99.20 249 0.07 99.60 244 0.18 97.6
B
Ethambutol — — — — — — — — — —
Rifampicin 120 118 0.16 98.33 125 0.12 104.2 115 0.18 95.83

The averaged concentration of APIs in each sample

tablet and the RSDs (< 1% for each tablet using both
wavelength channels, as shown in Tables 9 and 10)
showed good reproducibility of the method and injection.
The amounts of each API were compared to the labeled
value; for each API for each tablet of Sample A, the
amount was between 90 and 110%. The USP monograph
for this product specifies that there should be not less than
(NLT) 90% and not more than (NMT) 110% of the API in
the drug product. The assay demonstrated that each tablet
met the USP criteria. The three-API product, Sample B,
also passed the NLT 90% and NMT 110% criteria.

Application Note 257 7

 To evaluate method accuracy in another manner, the
Column: Acclaim PA2 3 µm, 4.6 × 150 mm
recoveries of APIs added to the sample placebos were Eluent: A: 8% CH3CN in 20 mM NaH2PO4
(plus 1.5 mL of TEA), pH 6.8
determined. Sample placebos without added APIs were 150 B: 50% CH3CN in 20 mM NaH2PO4
UV_vis_1
also prepared and analyzed with the HPLC method and UV_vis_2
(plus 1.5 mL of TEA), pH 6.8
Flow Rate: 1.0 mL/min
no interfering compounds were observed (Figure 4). Gradient: 100% A from -5 to 3 min, ramp to 100% B
in 0.5 min and hold 100% B for 7 min
The same amounts of APIs were added to the placebo Temperature: 35 °C
as shown on the sample label for the drug products. Inj. Volume: 5 µL
Detection: UV_vis_1 at 200 nm and 337 nm at 5 min
The spiked placebo samples were prepared and three UV_vis_2 at 238 nm
Sample: Sample A placebo
injections were made for each sample. The averaged
found concentration in each spiked placebo sample Peaks: Conc. (mg/L)
mAU
was compared to calculated concentration to
determine recovery.

0 1.3 2.5 3.8 5 6.3 7.5 8.8 10.5

Minutes 27673

Figure 4. Example chromatograms of Sample A Placebo

(chromatograms for Sample B Placebo were equivalent to
Sample A Placebo).

8 HPLC Assay Method for Drug Products Containing Anti-Tuberculosis Active Pharmaceutical Ingredients
 Table 11. Recovery at UV-vis_1
Calculated Spiked Average Found
Sample Compound RSD Recovery
Concentration (mg/L) Concentration (mg/L)
Isonaizid 57 55.8 0.06 97.89
Spiked Sample A Pyrazinamide 300 299 0.18 99.67
Placebo Ethambutol 202 197 0.17 97.52
Rifampicin 109 107 0.05 98.17
Isonaizid 81 78.3 0.11 96.67
Spiked Sample B Pyrazinamide 250 250 0.06 100.0
Placebo Ethambutol — — — —
Rifampicin 116 110 0.12 94.83

Table 12. Recovery at UV-vis_2

Calculated Spiked Average Found
Sample Compound RSD Recovery
Concentration (mg/L) Concentration (mg/L)
Isonaizid 57 55.8 0.05 97.89
Spiked Sample A Pyrazinamide 300 295 0.08 98.33
Placebo Ethambutol 202 — — —
Rifampicin 109 107 0.10 98.17
Isonaizid 81 78.4 0.06 96.79
Spiked Sample B Pyrazinamide 250 248 0.09 99.20
Placebo Ethambutol — — — —
Rifampicin 116 109 0.13 93.97

The recoveries in Spiked Placebo Sample A were

97.52 to 99.67% at UV-vis_1 and 97.89 to 98.33% at
UV-vis_2, and recoveries in Spiked Placebo Sample B
were 94.83 to 100% at UV-vis_1 and 93.97 to 99.20%
at UV-vis_2 (Tables 11 and 12). This experiment also
indicated that the single HPLC method was accurate for
all four APIs.

Application Note 257 9

Faster Analysis
This method can be made faster by using a smaller 800
UV_vis_1 Column: Acclaim RSLC PA2 2.2 µm, 2.1 × 100 mm
UV_vis_2 Eluent: A: 4% CH3CN in 20 mM NaH2PO4
column format and a smaller particle size. In this work (plus 1.5 mL of TEA), pH 6.8
B: 50% CH3CN in 20 mM NaH2PO4
(using the Acclaim RSLC PA2, 2.2 µm, 2.1 × 100 mm 2
(plus 1.5 mL of TEA), pH 6.8
column), faster separation was complete in less than Flow Rate: 1.0 mL/min
Gradient: 100% A from -2.5 to 0.5 min,
2 min with a system backpressure of ~ 570 bar. Figure 5 ramp to 100% B in 0.1 min and
hold 100% B for 1.2 min
shows a chromatogram of faster separation of the four-API Temperature: 35 °C
standard. Figures 6 and 7 show that faster separation also Inj. Volume: 1.5 µL
Detection: UV_vis_1 at 200 nm and 337 nm at 1 min
successfully analyzed the samples. UV_vis_2 at 238 nm
Sample: Sample A

Peaks: 1. Isoniazid
mAU 2. Pyrazinamide
Column: Acclaim RSLC PA2 2.2 µm, 2.1 × 100 mm 3. Ethambutol
Eluent: A: 4% CH3CN in 20 mM NaH2PO4 4. Rifampicin
(plus 1.5 mL of TEA), pH 6.8
B: 50% CH3CN in 20 mM NaH2PO4
(plus 1.5 mL of TEA), pH 6.8
900 Flow Rate: 1.0 mL/min 4
UV_vis_1
UV_vis_2 Gradient: 100% A from -2.5 to 0.5 min,
1
ramp to 100% B in 0.1 min and
2 hold 100% B for 1.2 min
Temperature: 35 °C
Inj. Volume: 1.5 µL
Detection: UV_vis_1 at 200 nm and 337 nm at 1 min 3
UV_vis_2 at 238 nm
Sample: Mixture of four anti-tuberculosis
drugs standard
4
Peaks: 1. Isoniazid
0 0.2 0.6 1 1.4 1.8
2. Pyrazinamide
3. Ethambutol Minutes 27675
mAU 4. Rifampicin
Figure 6. Faster separation of Sample A.

700 Column: Acclaim RSLC PA2 2.2 µm, 2.1 × 100 mm

1 Eluent: A: 4% CH3CN in 20 mM NaH2PO4
(plus 1.5 mL of TEA), pH 6.8
B: 50% CH3CN in 20 mM NaH2PO4
2 (plus 1.5 mL of TEA), pH 6.8
Flow Rate: 1.0 mL/min
3 Gradient: 100% A from -2.5 to 0.5 min,
ramp to 100% B in 0.1 min and
hold 100% B for 1.2 min
Temperature: 35 °C
0 0.2 0.6 1 1.4 1.8 Inj. Volume: 1.5 µL
Detection: UV_vis_1 at 200 nm and 337 nm at 1 min
Minutes 27674
UV_vis_2 at 238 nm
Sample: Sample A
Figure 5. Faster separation of a standard containing four
anti-tuberculosis drugs. mAU Peaks: 1. Isoniazid
2. Pyrazinamide
3. Rifampicin
1
3

0 0.2 0.6 1 1.4 1.8

Minutes 27676

Figure 7. Faster separation of Sample B.

10 HPLC Assay Method for Drug Products Containing Anti-Tuberculosis Active Pharmaceutical Ingredients
 Table 13. Peak Purity, Resolution, and Peak Analysis Results
for the Standard (UHPLC)
Resolution* Peak Purity RSD Peak Peak Purity RSD Peak Plates
Compound Asymmetry
(USP) Match Purity Match Index Purity Index (USP)
Isoniazid 2.37 1000 0.12 216.8 0.06 1.00 2775
Pyrazinamide 2.61 1000 0.24 228.5 0.11 0.94 3226
Ethambutol 14.82 989 4.25 196.3 1.97 1.42 1573
Rifampicin n.a. 1000 0.32 284.9 0.11 0.93 —

Table 14. Peak Purity, Resolution, and Peak Analysis Results for the Samples (UHPLC)
Sample Compound Peak Purity RSD Peak Peak Purity RSD Peak Resolution* Asymmetry Plates Match with
Match Purity Index Purity (USP) (USP) Library
Match Index
Isoniazid 1000 0.13 216.8 0.06 2.36 1.01 2726 1000
Pyrazinamide 1000 0.24 228.5 0.11 2.65 0.93 3156 1000
Sample A
Ethambutol 989 2.56 195.7 1.02 15.17 1.38 1701 999
Rifampicin 1000 0.27 284.8 0.09 n.a. 0.93 — 1000
Isoniazid 1000 0.13 216.8 0.06 2.33 1.02 2610 1000
Pyrazinamide 1000 0.25 228.6 0.11 24.79 0.94 3084 1000
Sample B
Ethambutol — — — — — — — —
Rifampicin 1000 0.39 285.0 0.14 n.a. 0.93 — 1000
*Calculation is based on USP and is compared to the next main peak.

The peak purity and spectral match data for the in the sample placebos and measuring the recovery;
standard and samples are displayed in Tables 13 and 14 there were good recoveries for both samples. The analysis
(spectra from the standard compounds were added to time can be accelerated with the faster method. This
the library for sample analysis) and support the visual separation was complete in < 2 min, providing high
observation from Figures 6 and 7 that a successful analysis throughput sample analysis.
was achieved with the fast method.
REFERENCES
Conclusion 1. http://en.wikipedia.org/wiki/Tuberculosis_treatment,
This work shows that a single HPLC method can accessed September 30, 2010.
be used to assay the four APIs in a combination drug 2. United States Pharmacopeia, 32 NF 27, 2009.
tablet used to treat TB. This 10 min method saves time
as well as mobile phase, compared to the two HPLC
assay methods described in the USP monograph for
this product. The analysis of two drug products yielded
acceptable percentage contents, as judged by the limits
in the USP monograph for the four-API drug product.
Method accuracy was also tested by spiking standards

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