This work was supported by the Korea Ministry of Environment (MOE) as part of the "The advancement of scientific research and technological development in environmental science program (E315-00015-0414-2)."

1. Preparation of Tubes in Administration Sets
NOTE: Precisely perform the cutting step to eliminate the effect of differences in tube length on drug sorption.
<ol>
	<li>Label the end of the tubes with the tube type (e.g., PVC, polyurethane (PU), and polyolefin (PO)) using a marker.</li>
	<li>Remove all detachable accessories, such as connectors and needle covers.</li>
	<li>Using a sharp razor to ensure clean edges, cut the tubes to 1 m in length from the connection of the drip chamber.</li>
</ol>
2. Dilution of Drug Injections
NOTE: Use a glass bottle (1 L) as the container for the injected drug solution. Perform the dilution step precisely. Verify the composition of the marketed drug product and use the same batch number for a whole experimental set.
<ol>
	<li>Label bottles with the drug names (e.g., diazepam or tacrolimus).</li>
	<li>Dilute the drug injections with 5% dextrose solution, from 5 mg/mL to 100 &#181;g/mL for diazepam injections (2 mL of diazepam injection in 100 mL of 5% dextrose solution) and from 5 mg/mL to 10 &#181;g/mL for tacrolimus injections (200 &#181;L of tacrolimus injection in 100 mL of 5% dextrose solution). 
	NOTE: Set up the tested concentrations of drugs and solvents based on the clinical usage.</li>
	<li>Gently mix the diluted solutions in the bottle by swirling to obtain homogenous drug solutions.</li>
	<li>Collect diluted solutions (1-10 mL) in amber vials using a glass graduated cylinder.</li>
	<li>Verify the concentration, as described in step 4. 
	NOTE: Use these concentrations of drugs as concentrations at the starting points.</li>
</ol>
3. Kinetic Sorption Study Using an Infusion Pump
NOTE: Confirm the tube-dependent flow rate using a pump prior to the sorption test due to the hardness of tubes. Collect samples at precise time points and use glass bottles and vials to prevent additional drug sorption during storage. Perform the test as shown in Figure 2. Protect the drug solution against light if the drug has photosensitivity. Perform the experiments in triplicate.
<ol>
	<li>Without creating air bubbles, preload a diluted solution of the drug into the tube using a syringe.
	<ol>
		<li>Connect one end of the tube to a syringe.</li>
		<li>Put the other end of the tube into the bottled drug solution.</li>
		<li>Pull back the syringe plunger until the tube is completely filled with the drug solution.</li>
	</ol>
	</li>
	<li>Install the tube into an infusion pump.
	<ol>
		<li>Open the door of the infusion pump and push the release lever.</li>
		<li>Insert the preloaded tube into the infusion pump and keep it straight.</li>
		<li>Remove the syringe at the end of the tube after installation.</li>
		<li>Put the end of tube into a chemically resistant borosilicate glass graduated cylinder to collect the drug solution after it passes through the tube.</li>
	</ol>
	</li>
	<li>Set the flow rate based on the type of tube in the administration set (PVC, PU, or PO) and the drug (e.g., 1 mL/min for diazepam and 10 mL/h for tacrolimus).</li>
	<li>Collect samples into amber vials at various time points, at room temperature.
	<ol>
		<li>Collect 1-mL diazepam samples at 0.05, 0.30, 0.55, and 1.05 h.</li>
		<li>Collect 10-mL tacrolimus samples at 1.05, 2.05, 3.05, and 4.05 h.</li>
	</ol>
	</li>
</ol>
4. Analysis of Drug Using High-performance Liquid Chromatography (HPLC)
NOTE: Recommended HPLC methods for drug analysis are described in references 1, 8,9. Use tandem mass spectrometry (MS/MS) and immunoassay after sample preparation as alternative methods10, 11. Perform the experiments in triplicate.
<ol>
	<li>Weigh the drugs and dissolve them in organic solvents at a concentration of 1 mg/mL as stock solutions.
	<ol>
		<li>Use methanol as a solvent for diazepam stock solution due to the low solubility of diazepam in 5% dextrose.</li>
		<li>Use acetonitrile as a solvent for tacrolimus stock solution due to the low solubility of tacrolimus in 5% dextrose.</li>
	</ol>
	</li>
	<li>Prepare standard solutions by diluting the stock solutions.
	<ol>
		<li>Dilute diazepam stock solutions with methanol to 0.3125, 0.625, 1.25, 2.5, 5.0, 10.0, and 20.0 &#181;g/mL.</li>
		<li>Dilute tacrolimus stock solutions with 5% dextrose to 2.5, 5.0, 10.0, 15.0, and 20.0 &#181;g/mL.</li>
	</ol>
	</li>
	<li>Analyze standards using the HPLC method with UV detection1, 8, 9. 
	NOTE: Use an appropriate detection method (UV, fluorescence, etc.) for drug analysis.
	<ol>
		<li>Inject 10 &#181;L of standards into an HPLC system with UV detection equipped with a C18 column (1.5 mm &#215; 250 mm, 5 &#181;m). See Table 1 for analysis conditions.</li>
		<li>Confirm the specificity and linearity1, 8, 9.
		<ol>
			<li>For specificity, monitor the drug peak (i.e., whether it was separated from other peaks in the chromatogram) to identify the drug.</li>
			<li>Confirm linearity at the calibration range (i.e., whether the peak area results are directly proportional to the concentrations).</li>
		</ol>
		</li>
		<li>Obtain calibration curves.
		<ol>
			<li>Create graphs based on concentration versus peak area value from chromatograms of the standards1, 8, 9.</li>
			<li>Obtain linear regression equations with R2 for the calibration curves (e.g., y = ax + b, x: concentration of drug, y: peak area)1, 8, 9.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Dilute the samples from the sorption study with methanol for diazepam or 5% dextrose for tacrolimus as appropriate so that they fall within the calibration range and directly inject 10 &#181;L of diluted sample into the HPLC system.</li>
</ol>
5. Calculation of Drug Concentration and Sorption Level
<ol>
	<li>Calculate the drug concentrations of the samples using calibration curves (unknown x and known y).</li>
	<li>Calculate the sorption levels of the drugs using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/55086/55086eq1.jpg" /> 
	where S: Sorption level (%) 
	Co: Drug concentration after the dilution of the injections (&#181;g/mL) 
	Cp: Drug concentration passed through the tubes (&#181;g/mL)</li>
</ol>

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A., Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+compatibility+with+new+polyolefin+infusion+solution+containers.">Drug compatibility with new polyolefin infusion solution containers.</a> Am. J. Health Syst. Pharm. 63, 2379-2382 (2006).</li><li>Cloyd, J. C., Vezeau, C., Miller, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Availability+of+diazepam+from+plastic+containers.">Availability of diazepam from plastic containers.</a> Am. J. Hosp. 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The authors have nothing to disclose.

Drug sorption to administration sets is a cause of unexpected drug loss in intravenous drug delivery. During sorption, drugs are generally partitioned to polymeric materials of tubes at the early phase of infusion; after sorption equilibrium is reached, the delivered amount of drug is stabilized1. The sorption levels of drugs should be evaluated and minimized. Several evaluation methods for drug sorption have been studied, such as a pump method and a drip method. Compared to the drip method, the pump method can be easily manipulated without bias. Although administration sets with flow regulators (conventional form) are used in the drip method, desired flow rates less than 5 mL/h are hard to achieve. Therefore, we recommend the pump method for the sorption evaluation of tubes in administration sets.
When using the pump method, major factors that affect drug sorption to tubes of administration sets are drug properties (e.g., hydrophobicity and charge), conditions of the sorption test (e.g., drug concentration, flow rate, solvent compatibility, tube length, and temperature), analytical methods for drugs (e.g., HPLC and MS), and the tube polymers in the administration sets (e.g., PVC, PU, and PO)2,3,4,5,6,7,8,9,10,11,12. First, the selection of the model drugs is critical for obtaining precise and accurate experimental results. Even though diazepam management is tracked by the Psychotropic Drugs Control Act, we selected diazepam (Figure 1a) and tacrolimus (Figure 1b) as model drugs because of their high sorption levels to polymeric tubes of administration sets1 or containers13, 14. In this case, drugs at high concentrations showed less sorption than those at low concentrations in the early phase of infusion1, 2. These drugs have high log P values (diazepam: 2.8215, tacrolimus: 3.9616) and low solubility, as categorized by the Biopharmaceutical Classification System (BCS class 2). Because of their hydrophobicity, these drugs can interact with tubes of administration sets, leading to sorption. Other drugs showing high sorption levels (e.g., nitroglycerin2 and cyclosporin A3) can be used as alternative model drugs for sorption evaluation. Furthermore, macromolecular drugs, such as biologics (antibody therapeutics, insulin, etc.), can be applied for the quality evaluation of administration sets regarding drug sorption12.
We set up a simple kinetic sorption study, using a pump to easily obtain precise results and to minimize artifacts (Figure 2). In the pump method, drug solution (Figure 2a) was passed through a tube cut from the administration set (Figure 2b) after installation into an infusion pump (Figure 2c). Except for the tubes from administration sets, all devices (bottle, graduated cylinder, and sampling vials) were composed of chemically resistant borosilicate glass to prevent additional drug sorption to polymers. In this study, tubes without other accessories at a fixed length of 1 m were used to simplify the factors of drug sorption to tubes of administration sets. If a clinical condition requires it, a multiplication factor for the length of tube can be used. In the sorption test, the diluted drug solutions were used as the starting concentrations1,7. After delivery, the drug solution (Figure 2d) was collected into vials at various time points (Figure 2e). Drug solutions for sampling were passed completely through the tube at the preselected conditions of flow rate and sampling time points. Sorption generally occurs in the early phase of infusion, and the pattern is followed by a convection-interfacial resistance-diffusion model7. Diazepam sorption results are comparable to the double-lumen extension tube model17 when the initial drug concentration is considered before delivery. Therefore, sampling time points can be modified so that sorption evaluation takes less time. All factors of the test conditions were confirmed based on the clinical usage of drugs.
In this protocol, we chose the HPLC method for drug analysis based on previous reports1, 8, 9. Simple and reproducible HPLC methods have been developed. The HPLC conditions are listed in Table 1. Various other techniques, such as MS and immunoassay, have also been developed as alternative analysis methods of drug concentrations10, 11. MS/MS and immunoassay are highly sensitive for the detection of drugs and their metabolites. Specifically, an immunoassay can easily be performed without requiring large and expensive equipment for drug analysis.
Regarding the quality evaluation of administration sets, drug sorption to PVC- and non-PVC-based materials used in the tubes of administration sets has been studied. The evaluation of sorption to tubes in administration sets started with drug selection and ended with the consideration of acceptable criteria of sorption levels, as illustrated (Figure 3). PVC-based tubes showed high sorption levels for many drugs such as diazepam, tacrolimus (Table 2), nitroglycerin2, and cyclosporin A3. Among approaches to minimize drug sorption to the tubes of administration sets to less than 10%, alternative materials or polymeric combinations have been developed, such as PO-based materials and layer-by-layer designs2, 13. The PE/PB/PP blended PO-based tube of an administration set used in this study showed low sorption levels, as a non-PVC-based tube. On the other hand, PE-based tubes are not used for administration sets, but they are commercially used in the market as a syringe extension tube due to their hardness.
This protocol can be applied to the quality control of administration sets with respect to drug sorption. More drugs classified by sorption level (highest, lower, and lowest) should be used in sorption evaluations for the quality assurance of administration sets. This protocol can also be used in scientific research for the development of new alternative polymeric materials or new designs for tubes in administration sets that do not result in drug sorption1, 13.

Administration sets are composed of a spike, a drip chamber, tubes, Luer adapters (connectors), and a needle cover for protection. Other accessories, such as an airway check valve, a regulating clamp, an in-line filter, a Y-tube with cap (an injection port), and a needle, can also be attached to administration sets. Drug sorption to tubes is a critical issue in the delivery of injectable drugs1. Sorption describes the adsorption of a drug to the polymer surface and the absorption of a drug into the polymeric matrix2. Drug sorption to tubes in administration sets causes unpredictable drug loss and makes it difficult to control the delivered drug concentration. Drug sorption to polymeric tubes is therefore a major impediment to the precise transfer of injectable drugs into the body. However, there are no standard methods or regulatory guidelines for the evaluation of drug sorption to tubes in administration sets.
The levels of drug sorption to tubes in administration sets have been reported using various evaluation methods1,2,3,4,5. Test methods and model drugs are key factors in sorption evaluation. The pump method1,3 and drip method4,5 have been widely used for sorption tests. In general, the pump method should be used in the case of drugs with low concentrations and low flow rates as the infusion conditions. Using various evaluation methods, many studies of drug sorption have been reported for polyvinyl chloride (PVC)- and non-PVC-based tubes in administration sets1,2,3,5. Many sorptive drugs can be selected to investigate whether the tubes of the administration sets have drug sorption potential or not. Diazepam (Figure 1a)1, 2, tacrolimus (Figure 1b)5, nitroglycerin2, and cyclosporin A3 are representative drugs with high sorption in PVC- and non-PVC-based tubes.
For the evaluation of drug sorption to the tubes, test conditions such as flow rate and drug concentration are based on the clinical use of the selected drugs1, 6, 7. In the case of diazepam, a high concentration of 100 &#181;g/mL was used at a flow rate of 1 mL/min to mimic the initial dose for the treatment of status epilepticus1. For tacrolimus, a concentration of 10 &#181;g/mL was delivered at a flow rate of 10 mL/h. Dextrose solution (5%) was used for the dilution of drug injections, and tube length was fixed at 1 m. Glass bottles and vials should be used to prevent additional sorption during the experiment and storage.
In this study, we conducted a kinetic sorption study with the model drugs, diazepam and tacrolimus, using a pump method. Specific details of this protocol, from tube preparation to sorption evaluation, were described previously. Methods for the evaluation of drug sorption have already been used to confirm drug properties for injections and to recommend the clinical use of injections with administration sets on a case-by-case basis1,2,3,4,5,6,7. This protocol may be used as a standard technique for the sorption evaluation of administration sets. International standards for the evaluation of drug sorption to tubes may be necessary to ensure the safety and efficacy of drug delivery.

<table>
	<tbody>
		<tr>
			<td>PVC IV sets</td>
			<td>Becton Dickinson 
			(BD) Co. Ltd. (Franklin Lakes, NJ, USA)</td>
			<td></td>
			<td>Internal diameter: 2.54 mm</td>
		</tr>
		<tr>
			<td>PU IV sets</td>
			<td>Tianjin Hanaco Medical (THM) Co.Ltd. 
			(Tianjin, China)</td>
			<td></td>
			<td>Non-PVC 
			Internal diameter: 2.54 mm</td>
		</tr>
		<tr>
			<td>Non-PVC Polyolefin IV sets</td>
			<td>Polyscientech, Co. Ltd (Anseong, Korea)</td>
			<td></td>
			<td>Non-PVC [PE elastomer/PP elastomer/PB elastomer(25/50/25, weight ratio) blend] 
			Internal diameter: 2.54 mm</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Korea Vaccine Co. Ltd. (Seoul, Korea)</td>
			<td>KOVAX-SYRINGE 1 mL 26G 1/2&#34;</td>
			<td></td>
		</tr>
		<tr>
			<td>Daewon diazepam injection</td>
			<td>Daewon Pharma. Co. Ltd. (Hwaseong, Gyunggi, Korea)</td>
			<td></td>
			<td>1 mg/mL, total 2 mL 
			Batch No.: P003 
			Composition: diazepam, propylene glycol, ethanol, benzyl alcohol, sodium benzoate, bezoic acid, water for injection</td>
		</tr>
		<tr>
			<td>Tacrobel injection</td>
			<td>Chong Keun Dang, Co. Ltd. (Seoul, Korea)</td>
			<td></td>
			<td>5 mg/mL, total 1 mL 
			Batch No.: AG002 
			Composition: tacrolimus hydrate, polyoxyl 60 hydrogenated castor oil (HCO-60), dehydrated alcohol</td>
		</tr>
		<tr>
			<td>5% Dextrose</td>
			<td>JW Pharmaceutical (Seoul, Korea)</td>
			<td>500 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Dextrose</td>
			<td>Daehan Pharmaceutical (Seoul, Korea)</td>
			<td>200 mL</td>
			<td>Bottle (glass)</td>
		</tr>
		<tr>
			<td>Amber vials</td>
			<td></td>
			<td>20 mL</td>
			<td>Glass</td>
		</tr>
		<tr>
			<td>Terumo infusion pump</td>
			<td>Terumo (Medical Corp., USA)</td>
			<td>TE-135</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC system with UV detector</td>
			<td>Agilent (Santa Clara, CA, USA)</td>
			<td>Agilent 1260</td>
			<td></td>
		</tr>
		<tr>
			<td>CAPCELL PAK C18 column&#160;</td>
			<td>Shiseido (Japan)</td>
			<td>90404</td>
			<td>1.5 mm x 250 mm, 5 &#956;m</td>
		</tr>
		<tr>
			<td>Diazepam</td>
			<td></td>
			<td></td>
			<td>From Daewon Pharma. Co., Ltd.</td>
		</tr>
		<tr>
			<td>Tacrolimus</td>
			<td>Teva Czech industries&#160; (Czech Republic)</td>
			<td></td>
			<td>From Chong Keun Dang, Co. Ltd.</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Burdick and Jackson Co., Ltd. (MI, USA)</td>
			<td>3/1/9017</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Burdick and Jackson Co. Ltd. (MI, USA)</td>
			<td>AH230-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Burdick and Jackson Co. Ltd. (MI, USA)</td>
			<td>3/1/4218</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid</td>
			<td>Sigma (St. Louis, MO, USA)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of drug sorption to pvc- and non-pvc-based tubes in administration sets using a pump

Administration sets are delivery tools for the direct application of drugs into the body and are composed of a spike, a drip chamber, tubes, Luer adapters (connectors), a needle cover for protection, and other accessories. Drug sorption to tubes of administration sets is a critical issue in terms of safety and efficacy. Although drug sorption is an important factor in the quality of an administration set, there are no standard evaluation methods for the regulation of drug sorption to the tubes. Here, we describe an evaluation protocol for drug sorption to tubes of administration sets. Tubes made of polyvinyl chloride (PVC)- and non-PVC-based polymeric materials were cut to 1 m in length. Diazepam and tacrolimus were used as model drugs. In the kinetic sorption study, we selected the drug concentration and flow rate based on the clinical usage of these drugs. After the dilution of each drug in a glass bottle, the diluted drug solution was delivered through tubes of administration sets using a pump. Samples were collected in amber vials at appropriate time points and the drugs were analyzed using high-performance liquid chromatography. Drug concentrations and sorption levels to tubes of the administration sets were calculated. Acceptable criteria to ensure the quality of administration sets are recommended.

The sorption to tubes in the administration set was kinetically monitored using the model drugs, diazepam (Figure 1a) and tacrolimus (Figure 1b), and the pump method (Figure 2). Diluted drug (Figure 2a) was passed through PVC- and non-PVC-based tubes (Figure 2b) at a fixed flow rate using an infusion pump (Figure 2c). The glass bottle was opened slightly to allow the insertion of the administration set tube. After the drug was delivered through the tubes (Figure 2d), samples were collected into amber vials (Figure 2e). All samples, including samples at the starting points, were analyzed using an HPLC method with UV detection (Figure 3a). Analysis conditions are listed in Table 1. For the preparation of drug standards, diazepam and tacrolimus were dissolved in methanol and acetonitrile because of their insolubility in 5% dextrose. The drug concentrations at the starting points of the sorption study were calculated from the analysis of the samples after the drug dilution. Sorption levels in PVC- and non-PVC-based tubes of administration sets were determined by calculating the percentage of remaining drug content after passage through the tubes from the calibration curves (Figure 3b) and subtracting these values from 100% (Figure 3c). The recommended acceptable range of drug sorption percentages was less than 10%, based on the content of injections from pharmacopoeias12. In addition, the specific drugs (e.g., anticancer drugs) should be confirmed with clinical guidelines. We determined whether drug sorption levels of the samples were appropriate (Figure 3d). Figure 4 and Figure 5 show representative chromatograms at low and high concentrations of drugs for standards and sample solutions, respectively. Retention time of each drug was 8.2 min for diazepam and 6.8 min for tacrolimus. There were no interfering peaks from the matrix. Specifically, in samples of the diazepam sorption study, the interfering peaks did not overlap with drug peak in the chromatogram, although a different solvent (5% dextrose) was used than in the standards. Table 2 shows calculations of representative sorption levels of diazepam and tacrolimus. The sorption level of each drug was the highest in PVC-based tubes, lower in PU-based tubes, and lowest in PO-based tubes at the initial phase of the kinetic sorption test.
<img alt="Figure 1" src="/files/ftp_upload/55086/55086fig1.jpg" /> 
Figure 1. Chemical structures of model drugs: (a) diazepam and (b) tacrolimus. Diazepam is a benzodiazepine derivative and tacrolimus is a 23-membered macrolide lactone. This figure has been modified1.
<img alt="Figure 2" src="/files/ftp_upload/55086/55086fig2.jpg" /> 
Figure 2. Test set of a kinetic sorption study using a pump. (a) Drug diluted with 5% dextrose in a bottle, (b) tube of administration set (1 m in length), (c) infusion pump, (d) drug passed through the tube, and (e) amber vials for storage. To minimize additional drug sorption, drug solutions and samples were prepared and stored in a glass bottle and amber vials for injections, respectively. This figure has been modified1.
<img alt="Figure 3" src="/files/ftp_upload/55086/55086fig3.jpg" /> 
Figure 3. Key steps for evaluating drug sorption level to tubes of administration sets. (a) Analysis of drug using an HPLC method with UV detection, calculation of (b) drug concentration from the calibration curve and (c) sorption level (%), and (d) recommendation of acceptable criteria for drug sorption. Several steps in the sorption evaluation, from drug selection to the sorption test, such as the analysis of drug concentration and the calculation of sorption levels, are described.
<img alt="Figure 4" src="/files/ftp_upload/55086/55086fig4.jpg" /> 
Figure 4. Representative chromatograms of diazepam. (a) Blank (methanol), standards at (b) 0.3125 &#181;g/mL and (c) 20 &#181;g/mL, and (d) sample (5% dextrose). The peak of diazepam was detected at 8.2 min, and fluctuations in retention time occurred within 1 min in the chromatograms. The peaks from the solvent were presented before the diazepam peak in the blank and standard chromatograms. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55086/55086fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55086/55086fig5.jpg" /> 
Figure 5. Representative chromatograms of tacrolimus. (a) Blank (acetonitrile with 5% dextrose), standards at (b) 2.5 &#181;g/mL and (c) 20 &#181;g/mL, and (d) sample. The tacrolimus peak at 6.8 min was successfully separated in a chromatogram. There were no peaks that overlapped with the tacrolimus peak. The peak was detected at 2.5 &#181;g/mL, with a signal-to-noise ratio greater than 10. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55086/55086fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">HPLC condition</td>
			<td align="center" colspan="2">Drug</td>
		</tr>
		<tr>
			<td>Diazepam</td>
			<td>Tacrolimus</td>
		</tr>
		<tr>
			<td>Mobile phase</td>
			<td>A mixture of acetonitrile, methanol, and sodium phosphate buffer* (29:47:24, v/v/v, adjusted to pH 3.1 with phosphoric acid) 
			*Sodium dihydrogen phosphate 1.2 g/L in distilled water</td>
			<td>Acetonitrile (100%)</td>
		</tr>
		<tr>
			<td>Flow rate</td>
			<td>0.1 mL/min</td>
			<td>0.1 mL/min</td>
		</tr>
		<tr>
			<td>UV wavelength</td>
			<td>232 nm</td>
			<td>213 nm</td>
		</tr>
		<tr>
			<td>Run time</td>
			<td>10 min</td>
			<td>10 min</td>
		</tr>
		<tr>
			<td>Retention time</td>
			<td>8.2 min</td>
			<td>6.8 min</td>
		</tr>
	</tbody>
</table>
Table 1. HPLC conditions.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Drug</td>
			<td colspan="3">Sorption (%)</td>
		</tr>
		<tr>
			<td>PVC</td>
			<td>PU</td>
			<td>PO</td>
		</tr>
		<tr>
			<td>Diazepam (0.05 h)</td>
			<td>27.6 &#177; 3.0</td>
			<td>21.9 &#177; 12.6</td>
			<td>11.3 &#177; 4.6</td>
		</tr>
		<tr>
			<td>Tacrolimus (1.05 h)</td>
			<td>15.1 &#177; 3.3</td>
			<td>10.3 &#177; 6.3</td>
			<td>0.6 &#177; 0.9</td>
		</tr>
	</tbody>
</table>
Table 2. Representative sorption results for diazepam and tacrolimus in PVC- and non-PVC-based tubes (n = 3)1.

Watch this Scientific Journal Video about Evaluation of Drug Sorption to PVC- and Non-PVC-based Tubes in Administration Sets Using a Pump at JoVE.com

Evaluation of Drug Sorption to PVC- and Non-PVC-based Tubes in Administration Sets Using a Pump

We report a method for drug sorption evaluation using a pump and model drugs (diazepam and tacrolimus). After drug analysis using high-performance liquid chromatography, drug concentrations and sorption levels in the tubes of administration sets are calculated.

Administration sets are delivery tools for the direct application of drugs into the body and are composed of a spike, a drip chamber, tubes, Luer adapters ...

evaluation-drug-sorption-to-pvc-non-pvc-based-tubes-administration

Nanyang Technological University

Research

JoVE Journal

Medicine

10.5K Views.  Yonsei University. We report a method for drug sorption evaluation using a pump and model drugs (diazepam and tacrolimus). After drug analysis using high-performance liquid chromatography, drug concentrations and sorption levels in the tubes of administration sets are calculated.

Video: Evaluation of Drug Sorption to PVC- and Non-PVC-based Tubes in Administration Sets Using a Pump

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically implanted catheters permit a researcher to effectively and humanely administer substances, perform repeated blood sampling and assists in conscious direct measurements of blood pressure and heart rate. Once the catheter is implanted long-term sampling is possible. Patency and catheter life depends on multiple factors including the lock solution used, flushing regimen and catheter material. This video will demonstrate the methodology of femoral artery and venous catheterization of the rat. In addition the video will demonstrate the use of the femoral venous and arterial catheters for blood sampling, drug administration and use of the arterial catheter in taking measurements of blood pressure and heart rate in a conscious freely-moving rat. A tether and harness attached to a swivel system will allow the animal to be housed and have samples taken by the researcher with minimal disruption to the animal. To maintain patency of the catheter, careful daily maintenance of the catheter is required using lock solution (100 U/ml heparinized saline), machine-ground blunt tip syringe needles and the use of syringe filters to minimize potential contamination. With careful aseptic surgical techniques, proper catheter materials and careful catheter maintenance techniques, it is possible to sustain patent catheters and healthy animals for long periods of time (several weeks).

Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

In multiple fields of study, access to the circulatory system in laboratory studies is necessary. Pharmacological studies in rats using chronically ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Evaluation of a Novel Laser-assisted Coronary Anastomotic Connector - the Trinity Clip - in a Porcine Off-pump Bypass Model

To simplify and facilitate beating heart (i.e., off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the Trinity Clip, is developed based on the excimer laser-assisted nonocclusive anastomosis technique. The Trinity Clip connector enables simplified, sutureless, and nonocclusive connection of the graft to the coronary artery, and an excimer laser catheter laser-punches the opening of the anastomosis. Consequently, owing to the complete nonocclusive anastomosis construction, coronary conditioning (i.e., occluding or shunting) is not necessary, in contrast to the conventional anastomotic technique, hence simplifying the off-pump bypass procedure. Prior to clinical application in coronary artery bypass grafting, the safety and quality of this novel connector will be evaluated in a long-term experimental porcine off-pump coronary artery bypass (OPCAB) study. In this paper, we describe how to evaluate the coronary anastomosis in the porcine OPCAB model using various techniques to assess its quality. Representative results are summarized and visually demonstrated.

This paper describes a novel nonocclusive coronary anastomotic connector in a porcine off-pump coronary artery bypass (OPCAB) model. This easy-to-use coronary connector has intrinsic potential to facilitate minimally invasive OPCAB surgery.

To simplify and facilitate beating heart (i.e., off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the ...

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 ...

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling molecules that affect multiple organs; they modulate gut motility and hormone production, and alter vascular tone, glucose metabolism, lipid metabolism, and energy utilization. Changes in fecal bile acids may alter the gut microbiome and promote colon pathology including cholerrheic diarrhea and colon cancer. Key regulators of fecal bile acid composition are the small intestinal Apical Sodium-dependent Bile Acid Transporter (ASBT) and fibroblast growth factor-19 (FGF19). Reduced expression and function of ASBT decreases intestinal bile acid up-take. Moreover, in vitro data suggest that some FDA-approved drugs inhibit ASBT function. Deficient FGF19 release increases hepatic bile acid synthesis and release into the intestines to levels that overwhelm ASBT. Either ASBT dysfunction or FGF19 deficiency increases fecal bile acids and may cause chronic diarrhea and promote colon neoplasia. Regrettably, tools to measure bile acid malabsorption and the actions of drugs on bile acid transport in vivo are limited. To understand the complex actions of bile acids, techniques are required that permit simultaneous monitoring of bile acids in the gut and metabolic tissues. This led us to conceive an innovative method to measure bile acid transport in live animals using a combination of proton (1H) and fluorine (19F) magnetic resonance imaging (MRI). Novel tracers for fluorine (19F)-based live animal MRI were created and tested, both in vitro and in vivo. Strengths of this approach include the lack of exposure to ionizing radiation and translational potential for clinical research and practice.

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport

Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice.

Along with their traditional role as detergents that facilitate fat absorption, emerging literature indicates that bile acids are potent signaling ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...

Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics stress-induced cardiomyopathy, the chronic model, administered through an osmotic pump, mimics advanced heart failure in humans. The purpose of the described protocol is to create the chronic ISO-induced heart failure model in mice using an implanted mini-pump. This protocol has been used to induce heart failure in 100+ strains of inbred mice. Techniques on surgical pump implantation are described in detail and may be relevant to anyone interested in creating a heart failure model in mice. In addition, the weekly cardiac remodeling changes based on echocardiographic parameters for each strain and expected time to model development are presented. In summary, the method is simple and reproducible. Continuous ISO administered via the implanted mini-pump over 3 to 4 weeks is sufficient to induce cardiac remodeling. Finally, the success for ISO model creation may be assessed in vivo by serial echocardiography demonstrating hypertrophy, ventricular dilation, and dysfunction.

The chronic administration of isoproterenol via an implanted osmotic pump has been used widely to mimic advanced heart failure in mice. Here, we describe detailed methods in surgical mini-pump implantation for the continuous isoproterenol administration over 3 weeks, as well as, echocardiographic assessment for the successful model creation.

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...