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 hair sample into an aluminum foil square and store at room temperature. Label the distal end of the hair to avoid possible contamination from additional handling of the proximal end.</li>
	<li>In addition to patient samples, collect &#8220;blank hair&#8221;: a scalp hair sample from someone who has not taken TB medication. Collect a large amount (&#62;30 mg blank hair for each 20 patient samples).</li>
</ol>
2. Drug extraction
<ol>
	<li>Label bead tubes. Each patient sample requires one tube. Label 12 tubes from &#8220;C0&#8221; to &#8220;C11&#8221;, one for each of the 12 calibration points. Label a tube for Low Quality Control, a tube for Medium Quality Control, and a tube for High Quality Control. Lastly, label a tube for Matrix Blank.</li>
	<li>Open the aluminum foil square containing hair sample. If hair sample is longer than 3 cm, cut hair at 3 cm from the proximal end and use that proximal portion for analysis.</li>
	<li>Weigh 2 mg of the hair sample into a bead tube.</li>
	<li>Weigh 2 mg of blank hair into 16 additional bead tubes. These will be used as the calibration points, quality controls, and matrix blank. The tubes will follow the same extraction procedure as the patient samples, aside from being spiked with drug reference standards at levels indicated in steps 2.8 and 2.9.</li>
	<li>Place all of the bead tubes into the homogenizer. Run homogenizer at speed 6.95 m/s. Run for two cycles of 30 s each, with a 15 s rest period in between the two cycles.</li>
	<li>Make internal standard mix and add it to the samples.
	<ol>
		<li>Add ~40 mL of methanol to a 50 mL volumetric flask.</li>
		<li>In amber glass vials, make the mixes of the internal standards shown in Table 1, using methanol as the solvent. 
		NOTE: Methanol is very volatile. Leave all vials capped during this process to prevent loss due to evaporation.</li>
		<li>From those mixes, add the volume shown in Table 1 to the 50 mL volumetric flask. Then, fill the flask to 50 mL with methanol.</li>
		<li>Cap and mix the volumetric flask. Add 500 &#956;L of the mixture to each of the pulverized hair tubes, except for the matrix blank tube. Add 500 &#956;L methanol to the matrix blank tube.</li>
	</ol>
	</li>
	<li>Make reference standard mixes.
	<ol>
		<li>Constitute neat reference standards of the following drugs with methanol to get the concentration shown in the table in step 2.7.2. Only 1 mL of the following concentrations is necessary.</li>
		<li>Add 1768 &#956;L of methanol to a vial, and then add the amounts of reference standard listed in Table 2 to the same vial to get 2 mL final volume. Label this vial &#8220;Ref Std Mix 1x&#8221;. Vortex.</li>
		<li>Spike 100 &#956;L from &#8220;Ref Std Mix 1x&#8221; into 900 &#956;L methanol in a new vial. Label this vial &#8220;Ref Std Mix 10x&#8221;. Vortex.</li>
		<li>Spike 100 &#956;L from &#8220;Ref Std Mix 10x&#8221; into 900 &#956;L methanol in a new vial. Label this vial &#8220;Ref Std Mix 100x&#8221;. Vortex.</li>
		<li>Spike 100 &#956;L from &#8220;Ref Std Mix 100x&#8221; into 900 &#956;L methanol in a new vial. Label this vial &#8220;Ref Std Mix 1000x&#8221;. Vortex.</li>
	</ol>
	</li>
	<li>Spike the calibration curve tubes by adding the amount of Ref Std Mix described in Table 3.</li>
	<li>Create QC mixes and spike quality control tubes.
	<ol>
		<li>Label 5 vials &#8220;QC-A&#8221; through &#8220;QC-E&#8221;.</li>
		<li>Add the following amounts of methanol to the five labeled vials: 
		QC-A: 990 &#956;L 
		QC-B: 940 &#956;L 
		QC-C: 950 &#956;L 
		QC-D: 980 &#956;L 
		QC-E: 950 &#956;L 
		&#160;</li>
		<li>Using the 1 mg/mL drug stocks created in step 2.7.1, add 10 &#956;L to the specific vials listed below. For the BDQ and CLF stocks, which are at 0.5 mg/mL, add 20 &#956;L to the vials listed below. 
		QC-A: PTH 
		QC-B: EMB, CLF, BDQ, PTM 
		QC-C: INH, LFX, LZD, MFX, PZA 
		QC-D: PTH, EMB 
		QC-E: CLF, BDQ, PTM 
		 
		NOTE: Some drugs are present in multiple mixes.</li>
		<li>Label a vial as &#8220;QC-A df100&#8221;. Dilute 10 &#956;L of QC-A into 990 &#956;L methanol.</li>
		<li>Label a vial as &#8220;Low QC stock&#8221;. Add 1832 &#956;L methanol to this vial. Add the amounts of QC mixes detailed below: 
		QC-A df100: 80 &#956;L 
		QC-B: 8 &#956;L 
		QC-C: 80 &#956;L 
		&#160;</li>
		<li>Label a vial as &#8220;Mid QC stock&#8221;. Add 760 &#956;L methanol to this vial. Add the amounts of QC mixes detailed below: 
		QC-A df100: 800 &#956;L 
		QC-B: 40 &#956;L 
		QC-C: 400 &#956;L 
		&#160;</li>
		<li>Label a vial as &#8220;High QC stock&#8221;. Add 1376 &#956;L methanol to this vial. Add the amounts of QC mixes detailed below: 
		QC-D: 160 &#956;L 
		QC-E: 320 &#956;L 
		MFX, 1mg/mL stock: 16 &#956;L 
		INH, 1mg/mL stock: 32 &#956;L 
		LFX, 1mg/mL stock: 32 &#956;L 
		LZD, 1mg/mL stock: 32 &#956;L 
		PZA, 1mg/mL stock: 32 &#956;L 
		&#160;</li>
		<li>Spike 10 &#956;L Low QC stock into the Low QC bead tube.</li>
		<li>Spike 10 &#956;L Mid QC stock into the Mid QC bead tube.</li>
		<li>Spike 10 &#956;L High QC stock into the High QC bead tube.</li>
	</ol>
	</li>
	<li>Place all tubes in hot shaker for 2 h at 37 &#176;C. Shaking should be slow enough that the water does not splash up on to the tubes.</li>
	<li>Remove tubes from shaker. Transfer liquid from bead tubes into new microcentrifuge tubes. Label these microcentrifuge tubes in the same way.</li>
	<li>Add 500 &#956;L methanol to the old tubes. Cap and vortex.</li>
	<li>For a second time, transfer liquid from the bead tubes into the corresponding microcentrifuge tube. It is okay to transfer pulverized hair. This will eventually be centrifuged out.</li>
	<li>Centrifuge the microcentrifuge tubes for 10 min at 2,800 x g.</li>
	<li>Carefully remove the liquid and transfer it into new centrifuge tubes with corresponding labels. Be careful not to disturb or transfer the hair pellet.</li>
	<li>Evaporate the liquid in the centrifuge tubes to dryness at 32 &#176;C.</li>
	<li>Reconstitute the samples by adding 200 &#956;L of mobile phase A (HPLC-grade water with 1% formic acid) to the dry tubes. Vortex.</li>
	<li>Transfer the liquid to amber vials with 250 &#956;L inserts.</li>
</ol>
3. LC-MS/MS preparation
<ol>
	<li>Make one liter of mobile phase A (HPLC-grade water with 1% formic acid) by adding some HPLC-grade water to a one-liter volumetric flask. Then add 10 mL of &#62;95% formic acid to that flask, and then fill to the line with HPLC-grade water.
	<ol>
		<li>Make one liter of mobile phase B (acetonitrile with 0.1% formic acid) by adding some acetonitrile to a one-liter volumetric flask. Then add 1 mL of &#62;95% formic acid to that flask, and then fill to the line with acetonitrile.</li>
	</ol>
	</li>
	<li>Install a 2 x 100 mm column with 2.5 &#956;m particle size and 100 &#197; pore size with polar endcapped, ether-linked phenyl beads fully made of porous silica in the column compartment. Ensure that column also has manufacturer recommended guard cartridge installed.</li>
	<li>Open the data acquisition software and double-click Hardware Configuration. Highlight LCMS and click Activate Profile.
	<ol>
		<li>Click New Sub-Project, or, if other sub-projects already exist, click Copy Sub-Project. Name the sub-project.</li>
		<li>Click New Document. Double-click Acquisition Method. Click Mass Spec within the Acquisition method window.</li>
		<li>Change the Scan Type dropdown to MRM (MRM). Make sure Polarity is set to Positive.</li>
		<li>Click Import List and select the .csv file MDR-TB LCMS method transitions.csv that is included in Supplemental Materials.</li>
		<li>Scroll down and set the Duration to 16.751 min. The appropriate cycle time and number of cycles will auto-populate.</li>
		<li>In left sidebar, click Integrated Valco Valve. Make sure that position name for step 0 is A. In Total Time (min) column, type in 0.4 in the first row and 13 in the second row.</li>
		<li>In the Position column, set row one to B and row two to A.</li>
		<li>In left sidebar, click Binary Pump. Set the gradient and flow rate table according to Table 4.</li>
		<li>In left sidebar, click Autosampler. Change injection volume to 10 &#956;L. Click Temperature control enabled and set to 4 &#176;C.</li>
		<li>In left sidebar, click Column Compartment. Set both right and left temperatures to 50 &#176;C.</li>
		<li>Close and save method.</li>
	</ol>
	</li>
	<li>Create batch by clicking New Document and selecting Acquisition Batch. Type in a set name and select the newly created method from the dropdown bar.
	<ol>
		<li>In a spreadsheet, create a batch that follows this order: calibration curve, quality controls, patient samples, calibration curve, quality controls, patient samples, calibration curve, 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. Put at least eight solvent blank injections after injections of the calibration curve and high quality control vial in order to reduce analyte carryover. 
		NOTE: More solvent blank injections may need to be added depending on column age.</li>
		<li>In the column adjacent to the sample names, type in the appropriate autosampler position for the corresponding vial.</li>
		<li>Click Add Set. In the pop-up window, type in the number of samples in the batch.</li>
		<li>Copy and paste sample names and vial locations from the spreadsheet to the newly created batch.</li>
		<li>Go to the Submit tab. Click Submit button.</li>
	</ol>
	</li>
	<li>Equilibrate system by inserting solvent line A into mobile phase A and solvent line B into mobile phase B. Open the purge valve on the binary pump.
	<ol>
		<li>Set solvent composition to 50% B at 4 mL/min flow rate. Turn binary pump on.</li>
		<li>After 5 min, decrease flow to 0.3 mL/min. Close the purge valve. Check for any leaks.</li>
		<li>In the software, press Equilibrate on the top toolbar. Set time to &#62;5 min, press OK.</li>
		<li>After the instrument has equilibrated, the modules in the bottom right of the window will appear green. Check that pressure has stabilized, and then start the batch by clicking Start Sample.</li>
	</ol>
	</li>
</ol>
&#160;4. Data analysis
<ol>
	<li>After the batch is completed, open the quantitation software. Click the wand icon to create a new Results table.
	<ol>
		<li>Click Browse to navigate to the appropriate folder, and then highlight the data file and click the right-pointing arrow to move the data into the Selected area. Click Next.</li>
		<li>Select Create New Method and click New. Input new quantitation method name and press Save and then Next.</li>
		<li>Select the first injection of the middle calibration point. Press Next.</li>
		<li>Tick mark all the transitions of the internal standards in the IS column.</li>
		<li>For the quantifier transitions for the reference standards, select the corresponding IS in the IS Name column. Click Next.</li>
		<li>Scroll through the transitions to assure that the automatically selected retention time is accurate. Make sure that Gaussian Smoothing is set to 1.5. All other default settings can remain as is (i.e., Noise Percentage 100%, Baseline Sub. Window 2.00 min, Peak Splitting 2 points). 
		NOTE: If wanted, modify automatic integration parameters at this point. Since these parameters change based on instrument setup, we have not included ours here.</li>
		<li>Click Finish to apply the quantitation method to the batch.</li>
	</ol>
	</li>
	<li>Click the top left Displays the peak review button to view chromatograms. Navigate through the transitions using the left sidebar. Scroll through each injection of every quantifier transition and manually integrate the correct peak if necessary.
	<ol>
		<li>To manually integrate a peak, click on the Enable manual integration mode button, zoom into the chromatogram by clicking and dragging along the x- or y-axis, and then draw a line from one baseline to the other baseline, defining the peak. Figure 3 shows two chromatograms: one that has INH, and therefore has been manually integrated, and another that does not have INH. 
		NOTE: All injections must be integrated using the same parameters. Peak width can provide a guideline for adhering to these parameters, but sometimes peak width will differ. To quantify a peak, the retention time must be within &#177;0.15 min of the expected retention time for that analyte (as defined by the reference standard peaks), qualitatively confirmed as having the expected quantifier to qualifier ratio (as shown in Figure 2), and have a signal-to-noise ratio of greater than 10.</li>
	</ol>
	</li>
	<li>In the Sample Type column, set the calibration curve injections (with the exception of the blank calibration curve injections) to Standard. Set the quality control injections to Quality Control. Leave the remaining injections as Unknown. 
	NOTE: This will be set across all transitions.</li>
	<li>In the Actual Concentration column, type in the concentrations found in Table 5 for all calibration curve and quality control injections.</li>
	<li>Click the second from top left Displays the calibration curve button. Click the Regression button.</li>
	<li>Set Weighting Type to 1/x and press OK.</li>
	<li>Validate the calibration curve and quality control samples to assure that the batch ran successfully.
	<ol>
		<li>For each quantifier reference transition (not internal standard transitions), look at each calibration curve injection accuracy (in the Accuracy column). At least two-thirds of the calibration points must have an accuracy within 80-120%.</li>
		<li>For calibration points far outside of the expected accuracy, the injection may be an outlier. Exclude outliers if their calculated concentration is more than two standard deviations away from the other two injections of that vial. Clicking the &#8220;et peak to &#8216;not found button above each chromatogram.</li>
		<li>Check that the R-value displayed above the calibration curve is &#62;0.975.</li>
		<li>Check that all quality control injections have an accuracy within 80-120%.</li>
	</ol>
	</li>
	<li>If all above conditions are satisfied, the batch has passed, and samples can be quantified. Click Edit in the toolbar, then click Copy entire table. Paste the table in a spreadsheet.</li>
	<li>Take the average of the calculated concentration of the two sample injections to determine the reported concentration of each sample.</li>
</ol>

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This work was supported by the National Institute of Allergy and Infectious Diseases RO1 AI123024 (Co-PIs: John Metcalfe and Monica Gandhi).

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:755px;" width="755">
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	<tbody>
		<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>
		</tr>
		<tr height="21">
			<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>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">OPC 14714</td>
			<td style="width:211px;">Toronto Research Chemicals</td>
			<td>O667600</td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</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>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Turbovap LV evaporator</td>
			<td style="width:211px;">Biotage</td>
			<td style="width:161px;">103198/11</td>
			<td></td>
		</tr>
	</tbody>
</table>

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. ...

validated-lc-msms-panel-for-quantifying-11-drug-resistant-tb

Research

JoVE Journal

Medicine

7.5K Views.  University of California San Francisco. Current methods of analyzing patients’ 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.

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

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

A Novel Approach for the Administration of Medications and Fluids in Emergency Scenarios and Settings

The available routes of administration commonly used for medications and fluids in the acute care setting are generally limited to oral, intravenous, or intraosseous routes, but in many patients, particularly in the emergency or critical care settings, these routes are often unavailable or time-consuming to access. A novel device is now available that offers an easy route for administration of medications or fluids via rectal mucosal absorption (also referred to as proctoclysis in the case of fluid administration and subsequent absorption). Although originally intended for the palliative care market, the utility of this device in the emergency setting has recently been described. Specifically, reports of patients being treated for dehydration, alcohol withdrawal, vomiting, fever, myocardial infarction, hyperthyroidism, and cardiac arrest have shown success with administration of a wide variety of medications or fluids (including water, aspirin, lorazepam, ondansetron, acetaminophen, methimazole, and buspirone). Device placement is straightforward, and based on the observation of expected effects from the medication administrations, absorption is rapid. The rapidity of absorption kinetics are further demonstrated in a recent report of the measurement of phenobarbital pharmacokinetics. We describe here the placement and use of this device, and demonstrate methods of pharmacokinetic measurements of medications administered by this method.

This study presents a novel device that offers an easy route to quickly provide medications and fluids in patients with limited or difficult IV access. This small-diameter device is placed in the distal third of the rectum, allowing for ongoing medication and fluids administration.

The available routes of administration commonly used for medications and fluids in the acute care setting are generally limited to oral, intravenous, or ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Taste Exam: A Brief and Validated Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject&#39;s reported sensation and taste receptor genotype.

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, including humans. The advantage of hair cortisol concentration is that it reflects a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time, much like hemoglobin A1C represents glycemic control. However, obtaining hair samples can be challenging, due to the cultural beliefs and hair care practices of minority participants. For example, African Americans may be reluctant to provide samples. Additionally, few researchers are trained to collect hair samples from African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain hair samples from African Americans. To illustrate the representative results of this protocol implementation, de-identified data from African Americans that participated in a community-based study on chronic stress are provided. Hair practice preferences are assessed. The participants are made comfortable by showing pictures of hair samples prior to cutting their hair. The single strain twist and gently pull method is used to collect approximately 30 - 50 strands of hair from the posterior vertex region of the scalp. This protocol will significantly improve collection of hair samples from African Americans.

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.