Name: validated lc-ms/ms panel for quantifying 11 drug-resistant tb medications in small hair samples
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The authors would like to thank Professor Keertan Dheda, Dr. Ali Esmail, and Marietjie Pretorius at the University of Cape Town Lung Institute who facilitated the collection of hair samples for the study. The authors further gratefully acknowledge the contributions of the participants of this study.

All patients provided written informed consent prior to hair sample collection. We obtained Institutional Review Board approval from the University of Cape Town and the University of California, San Francisco.
1. Hair sampling
<ol>
	<li>Obtain written informed consent.</li>
	<li>Use clean scissors to cut approximately 20-30 scalp hair strands from the occipital region as close to the scalp as possible.</li>
	<li>Place tape around the distal side of the hair to indicate directionality. Fold h...

<ol>
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We report here the protocol for the method we developed and validated for quantifying 11 anti-TB medications utilized in the treatment of DR-TB in small hair samples using LC-MS/MS. No other method for quantifying these 11 drugs in hair has been previously developed, validated and published. Our method can quantify sub-nanogram levels of drugs in only 20-30 hair strands of approximately 3 centimeters (cm) in length (~2 mg) and has already been validated22. The low weight of hair analyzed means tha...

In the twenty-first century, drug-resistant TB (DR-TB) is an evolving catastrophe for already weak national TB control programs, with confirmed cases doubling in the past 5 years alone, accounting for nearly one-third of all deaths related to antimicrobial resistance globally1,2. Successful treatment of DR-TB has conventionally required longer and more toxic second-line regimens than treatment for drug-sensitive TB. Moreover, patients with DR-TB often have significant pre-existing challenges to adherence, which contributed to the emergence of resistance initially3.
Unlike HIV infection where viral loads can be used to monitor treatment, surrogate endpoints of treatment response in TB are delayed and unreliable on an individual level4. Monitoring patient adherence, an important predictor of subtherapeutic anti-TB drug concentration and treatment failure, is also challenging. Self-reported adherence suffers from recall bias and the desire to please providers5,6. Pill counts and medication event monitoring systems (MEMS) can be more objective7 but do not measure actual drug consumption8,9,10. Drug levels in biomatrices can provide both adherence and pharmacokinetic data. Therefore, plasma drug levels are commonly used in therapeutic drug monitoring11,12. In the context of drug adherence monitoring, however, plasma levels represent short-term exposure and are limited by significant intra- and inter-patient variability when determining appropriate adherence reference range. &#8220;White coat&#8221; effects, where adherence improves prior to clinic or study visits, further complicates the ability of plasma levels to provide accurate drug adherence patterns13.
Hair is an alternative biomatrix that can measure long-term drug exposure14,15. Many drugs and endogenous metabolites incorporate into the hair protein matrix from the systemic circulation as hair grows. As this dynamic process continues during hair growth, the amount of drug deposited in the hair matrix depends on the continuous presence of the drug in circulation, making hair an excellent temporal readout of drug intake. Hair as a biomatrix has the additional advantage of being easily collected without the need for cold chain for storage and shipment compared to blood. Moreover, hair is non-biohazardous, which provides additional feasibility advantages in the field.
Hair drug levels have long been used in forensic applications16. Over the last decade, hair antiretroviral (ARV) levels have demonstrated utility in assessing drug adherence in HIV treatment and prevention, to which our group contributed. ARV levels in hair have been shown to be the strongest independent predictors of treatment outcomes in HIV infection17,18,19,20,21. To determine whether hair levels of DR-TB patients will have the same utility in predicting treatment outcome, we used LC-MS/MS to develop and validate a method for analyzing 11 DR-TB medications in small hair samples. As an initial assessment of the assay&#8217;s performance, we measured DR-TB drugs levels in a convenience sample of patients with DR-TB receiving directly observed therapy (DOT) in the Western Cape, South Africa22.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2 mL injection vials</td>
			<td style="width:211px;">Agilent Technologies</td>
			<td style="width:161px;">5182-0716</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">250 uL injection vial inserts</td>
			<td style="width:211px;">Agilent Technologies</td>
			<td style="width:161px;">5181-8872</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bead ruptor 24</td>
			<td style="width:211px;">OMNI International</td>
			<td style="width:161px;">19001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bead ruptor tubes (2 mL bead kit, 2.8mm ceramic, 2 mL microtubes)</td>
			<td style="width:211px;">OMNI International</td>
			<td style="width:161px;">19628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bedaquiline</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">B119550</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Bedaquiline-d6</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">B119552</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Clofazimine</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>C324300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Clofazimine-d7</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>C324302</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable lime glass culture tubes</td>
			<td style="width:211px;">VWR</td>
			<td style="width:161px;">60825-425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethambutol</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>E889800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethambutol-d4</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>E889802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethionamide</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">E890420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethionamide-d5</td>
			<td style="width:211px;">ClearSynth</td>
			<td>CS-O-06597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formic acid</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:161px;">F0507-100mL</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Glass bottles</td>
			<td style="width:211px;">Corning</td>
			<td style="width:161px;">1395-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hot Shaker</td>
			<td style="width:211px;">Bellco Glass Inc</td>
			<td style="width:161px;">7746-32110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC</td>
			<td style="width:211px;">Agilent Technologies</td>
			<td style="width:161px;">Infinity 1260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC grade acetonitrile</td>
			<td style="width:211px;">Honeywell</td>
			<td style="width:161px;">015-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC grade methanol</td>
			<td style="width:211px;">Honeywell</td>
			<td style="width:161px;">230-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC grade water</td>
			<td style="width:211px;">Aqua Solutions Inc</td>
			<td style="width:161px;">W1089-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoniazid</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">I821450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isoniazid-d4</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>I821452</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LC column, Synergi 2.5 um Polar RP 100 A 100 x 2 mm</td>
			<td style="width:211px;">Phenomenex</td>
			<td style="width:161px;">00D-4371-B0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LC guard cartridge</td>
			<td style="width:211px;">Phenomenex</td>
			<td style="width:161px;">AJ0-8788</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LC guard cartridge holder</td>
			<td style="width:211px;">Phenomenex</td>
			<td style="width:161px;">AJ0-9000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LC-MS/MS quantitation software</td>
			<td style="width:211px;">Sciex</td>
			<td style="width:161px;">Multiquant 2.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Levofloxacin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td dir="LTR">1362103-200MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Levofloxacin-d8</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">L360002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Linezolid</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">L466500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Linezolid-d3</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>L466502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro centrifuge tubes</td>
			<td style="width:211px;">E&#38;K Scientific</td>
			<td style="width:161px;">695554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moxifloxacin</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>M745000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Moxifloxacin-13C, d3</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">M745003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS/MS</td>
			<td style="width:211px;">Sciex</td>
			<td style="width:161px;">Triple Quad 5500</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">OPC 14714</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>O667600</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pretomanid (PA-824)</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td style="width:161px;">P122500</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Prothionamide</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>P839100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Prothionamide-d5</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>P839102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pyrazinamide</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">P840600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pyrazinamide-15N, d3</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td dir="LTR">P840602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Septum caps for injection vials</td>
			<td style="width:211px;">Agilent Technologies</td>
			<td style="width:161px;">5185-5862</td>
			<td></td>
		</tr>
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validated lc-ms/ms panel for quantifying 11 drug-resistant tb medications in small hair samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

An illustration of a chromatogram with confirmed levels of all 11 DR-TB drugs is shown in Figure 1. The retention time for each analyte can change when using different instruments and columns, so the exact retention time should be determined individually.
The Extracted Ion Chromatograms (EICs) for one particular drug (isoniazid, INH) in one of the calibrators (blank hair sample spiked with DR-TB drug reference standards) are shown in Figure 2<...

Watch this Scientific Journal Video about Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples at JoVE.com

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

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...

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.

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