Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Patient adherence is very challenging and drug metabolism can vary person to person so objective measurement of drug levels may prove to be important in preventing the emergence of resistance during TB treatment. If adherence could be more accurately and easily quantified, then healthcare providers could develop a treatment plan that would limit the development of drug resistance. We have developed the first multiple analyte mass spectrometry panel for multiple TB medications within small hair samples in a single assay.

Our multi-drug panel only requires one two-milligram sample per patient and uses neither solid phase extraction or filtering step to prevent any loss of sample volume. Objective measures of drug exposure measured early in the treatment course could potentially alert clinicians to problems with individual drugs in the treatment regimen. Hair samples must be pulverized on the day of analysis for an accurate drug concentration level measurement.

To acquire hair samples, after obtaining written informed consent, get materials ready including a thin label, two participant labels, a pair of scissors, an unfolded piece of aluminum foil, an alcohol wipe and a plastic bag. Wipe the blades of the scissors clean with an alcohol wipe. Go to the back of the head and lift the top part of the hair using a hair clip if desired.

Gather 30 strands of hair from the back of the head and isolate the thatch with your hand. Take the scissors down to the scalp and cut the thatch of hair. Carefully place the thatch of hair on the aluminum foil.

Put the thin label over the distal end of the hair thatch, taping it securely to the aluminum foil. The distal end is the side furthest from the scalp. Fold over the piece of aluminum foil on top of the hair thatch and put a participant label on top of the aluminum foil.

Place the folded over piece of aluminum foil into the plastic bag and put another participant label on top of the plastic bag. For drug extraction from the hair samples, weigh out two milligrams of each hair sample into individually labeled bead tubes and two milligrams of the blank hair sample into each of 16 additional bead tubes. After weighing, place all of the bead tubes into a bead mill homogenizer and homogenize the samples at a speed of 6.95 meters per second for two 30-second cycles with a 15-second rest period between each cycle.

After the second homogenization, add 500 microliters of the appropriate internal standard mix to each sample and add 500 microliters of methanol to the matrix blank tube. Spike the calibration curve tubes with the appropriate volume of reference standard mix according to the table and place all of the tubes in a 37 degree water bath with gentle shaking for two hours. At the end of the incubation, transfer the supernatant from each bead tube into new labeled microcentrifuge tubes and add 500 microliters of methanol to the bead tubes.

Vortex the bead tubes and transfer the methanol wash to the corresponding microcentrifuge tubes. Centrifuge the samples and carefully transfer the supernatants into new tubes without disturbing the hair pellets. Evaporate the liquid in the tubes to dryness at 32 degrees Celsius and reconstitute the samples with 200 microliters of high pressure liquid chromatography grade water with 1%formic acid.

After vortexing, transfer the liquid samples to amber vials with 250 microliter inserts. For liquid chromatography tandem mass spectrometry, first install a two to 100 millimeter column with a 2.5 micrometer particle size and 100 angstrom pore size with polar-endcapped etherlinked phenol beads fully made of porous silica into the column compartment. Confirm that the column also has a manufacturer recommended guard cartridge installed.

Copy and paste the compound information included in the MDR-TB LCMS method transitions. csv file into the box in the mass spec window. In a spreadsheet, create a batch sequence of the calibration curve, quality controls, patient samples, calibration curve, quality controls, patient samples, calibration curve and quality controls.

Add solvent blank injections at the start and end of the run as well as before and after the calibration curve, quality controls and patient samples. Then place at least eight solvent blank injections in the injections in the calibration curve and the high quality control vial to reduce the analyte carryover. When the batches complete, open the quantitation software.

Scroll through the transitions to ensure that the automatically selected retention time is accurate and check that the Gaussian smoothing is set to 1.5. Next, click displays the calibration curve and regression and set the waiting type to one over x. Click OK and validate the calibration curve and quality control samples to assure that the batch ran successfully.

For each quantifier reference analyte, at least two-thirds of the calibration points must have an accuracy within 80-120%Check that the R value displayed above the calibration curve is greater than 0.975. If all of the above conditions are satisfied, the batch has passed and the samples can be quantified. Click Edit in the toolbar and click Copy Entire Table.

Then paste the table into a spreadsheet and use the average of the calculated concentration of the two sample injections to determine the reported concentration of each sample. Here is shown an illustration of a chromatogram with confirmed levels of all tested 11 drug-resistant TB drugs. In these figures, the extracted ion chromatograms for one particular drug in one of the calibrators can be observed.

The quantifier and qualifier transitions are used to qualitatively confirm the presence of the drug as the ratio between the area of the quantifier and the area of qualifier remains the constant across samples. The internal standard is also monitored to ensure that each sample injection is normalized. In this analysis, a convenient sample of 15 hair samples among a total study population of 96 patients taking drug-resistant TB drugs under directly observed therapy conditions from Western Cape, South Africa was evaluated to determine representative levels of drug-resistant TB drugs across the lowest and highest levels for each analyte.

In the absence of a solid phase extraction or filtering step, take care when transferring the supernatant into the evaporation tubes. Anything transferred into the tubes can end up injected into the column. As the rotor of the bead raptor can fly off if not tightly screwed or locked, be sure to double check the rotor before operating.

In contrast to antiretrovirals in the field of HIV, the relationship between TB medication pharmacokinetics, adherence and clinical outcomes remains largely unstudied. We have hypothesized that some TB medications do not accumulate well in hair. Targeting the metabolites of these drugs may provide a better understanding of patient adherence.