Page:NIOSH Manual of Analytical Methods - 8007.pdf/3

 TOLUENE in Blood: METHOD 8007, Issue 1, dated 5 March 2013 - Page 3 of 5

STOCK STANDARD PREPARATION: 9.	Prepare a primary stock standard solution by diluting 20 µL of toluene to 50 mL with ethylene glycol (20 µL/50 mL = 345.7 µg/mL.) Store in a tightly capped glass container having little or no head space. NOTE: 	Aliquots of toluene should be introduced below the ethylene glycol to prevent loss of toluene. 10.	Prepare stock standards by diluting aliquots of the primary standards with ethylene glycol. Suggested levels: 34.6 µg/mL, 3.46 µg/mL, 0.346 µg/mL. CALIBRATION AND QUALITY CONTROL: 11.	Calibrate with at least five working standards in duplicate covering the concentration range of the samples. a.	 Prepare each working standard by adding 1.5 mL of IS solution and 1.5 mL of blank blood to a headspace vial and cap the vial. Spike the mixture in the vial (through the septum using a micro-syringe) with a stock standard solution to the desired concentration of toluene and mix by shaking. b.	 Prepare at least two blanks by repeating step a. but omitting the toluene spike. c.	 Measure the peak areas of toluene and isobutanol in the chromatograms. Subtract the average toluene peak area of the blank from the toluene peak areas of the standards (see NOTE 1.) Divide the peak area of the blank-corrected toluene by the peak area from the isobutanol peak. Prepare a calibration curve of the peak area Std/area IS versus the toluene concentration of the standards. NOTE 1: 	A trace amount of toluene may be present in the blood of some donors. These levels will vary depending upon environmental exposures. If the blanks show the presence of toluene the standards need to be blank corrected, or else the sample results will be biased low. Geometric means for the U.S. population as determined from the National Health and Nutrition Examination Survey may be useful for comparison. [8] NOTE 2:	 It is also highly recommended that a reagent blank or blanks be included in the analysis. Atmospheric toluene in the lab may contribute errors to the measured values. A reagent blank using water, a blank headspace vial, or both of these options will show if the laboratory conditions are free from a quantifiable amount of toluene. 12.	Prepare two levels of quality control (QC) samples by spiking toluene into whole blood. These levels could be at approximately 10 times the LOQ and 200 times the LOQ, but could be adjusted to better suit the anticipated levels of the sample set. Unspiked samples of the blood used to prepare the QC samples should be analyzed to determine the blank level and the true target level. QC samples should be analyzed with every batch such that they constitute 10% of the sample batch. 13.	QC values should be within +/- 20% of the spiked values. If not, the batch is considered out of control, the batch data discarded, and corrective actions should be taken before more samples are analyzed. Alternatively, if the method has a long history in the lab allowing enough data for control charts to be constructed, the control charts could serve as the in or out of control decision guide, which could be looser or stricter than the recommended 20%. MEASUREMENT: 14.	Set the gas chromatograph according to manufacturer’s recommendations and to conditions given on page 8007-1. 15.	Set the headspace sampler according to manufacturer’s recommendations and to conditions given on page 8007-1. 16.	Inject and analyze samples, standards, QC samples, and blanks. 17.	Measure the peak area of both toluene and isobutanol in the chromatograms (do not subtract the blank from the samples). Divide the peak area of the toluene peak by the area from the isobutanol peak. NIOSH Manual of Analytical Methods (NMAM), Fifth Edition