Page:NIOSH Manual of Analytical Methods - 5032.pdf/3

 PENTAMIDINE ISETHIONATE: METHOD 5032, Issue 2, dated 15 August 1994 - Page 3 of 4 SAMPLE PREPARATION:

7. 8. 9. 10.

11.

NOTE: Sample preparation should be performed in a biological safety cabinet. Gloves should be worn. Place the 37-mm PVC filter with the exposed side facing upward into a 50-mL beaker. Add 3.0 mL of eluent to the beaker. Seal the mouth of the beaker with plastic, flexible, water-resistant film. Place beaker into ultrasonic bath for 10 min. Use disinfectant as medium in ultrasonic bath. Transfer solution to a sample vial with a disposable pipet. NOTE: Filtration of the sample solution is not recommended because fluorescent material could be leached from the filter or the housing for the filter, causing interference during analysis. Filtration is unnecessary because the guard column protects the analytical column from particulate matter. Recover pentamidine isethionate from the interior surface of the front piece of the cassette filter holder. a. Add 3.0 mL of eluent to this cassette piece (original plug remains in the inlet). b. Wet much (about 80%) of the interior surface by tilting the cassette piece. Avoid bringing eluent into contact with glove being worn. Complete this step in about 15 seconds to minimize evaporation. c. Transfer solution to a sample vial with a disposable pipet.

CALIBRATION AND QUALITY CONTROL: 12.

13.

14.

Calibrate daily with at least eight working standards over the range 2 to 1000 ng of pentamidine isethionate per mL of solution. a. Prepare a series of working standards in eluent by serial dilution. NOTE: Working standards in eluent are stable for more than 3 months when stored in the dark at 0 °C. b. Analyze together with samples and blanks (steps 15 through 17). c. Prepare a calibration graph (peak height or area versus concentration of pentamidine isethionate). Determine recovery (R) at least once for each lot of PVC membrane filters used for sampling in the calibration range (step 12). Prepare three filters at each of five levels plus three media blanks. a. Place filters into 50-mL beakers. b. By means of a 10-µL syringe, fortify each filter with recovery solution. c. Seal the mouth of each beaker with plastic, flexible, water-resistant film. d. Store each sample overnight in the dark at 25 °C. e. Prepare samples (steps 7 through 10) and analyze with working standards (steps 15 through 17). f. Prepare a graph of R vs. ng of pentamidine isethionate recovered. Analyze three quality control blind spikes and three analyst spikes to ensure that the calibration graph is in control.

MEASUREMENT: 15. 16. 17.

Set liquid chromatograph to manufacturer's recommendations and to conditions given on page 5032-1. Inject 100-µL sample aliquot manually or with autosampler. Measure peak area or peak height.

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 8/15/94