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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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&lt;br/&gt; (BD) Co. Ltd. (Franklin Lakes, NJ, USA)</td><td></td><td>Internal diameter: 2.54&amp;nbsp;mm</td></tr><tr><td>PU IV sets</td><td>Tianjin Hanaco Medical (THM) Co.Ltd.&lt;br/&gt; (Tianjin, China)</td><td></td><td>Non-PVC&lt;br/&gt;Internal diameter: 2.54&amp;nbsp;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]&lt;br/&gt;Internal diameter: 2.54&amp;nbsp;mm</td></tr><tr><td>Syringe</td><td>Korea Vaccine Co. Ltd. (Seoul, Korea)</td><td>KOVAX-SYRINGE 1 mL 26G 1/2&quot;</td><td></td></tr><tr><td>Daewon diazepam injection</td><td>Daewon Pharma. Co. Ltd. (Hwaseong, Gyunggi, Korea)</td><td></td><td>5 mg/mL, total 2 mL&lt;br/&gt; Batch No.: P003&lt;br/&gt; 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&lt;br/&gt; Batch No.: AG002&lt;br/&gt; 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 C&lt;sub&gt;18&lt;/sub&gt; column&amp;nbsp;</td><td>Shiseido (Japan)</td><td>90404</td><td>1.5 mm x 250 mm, 5 &amp;mu;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&amp;nbsp; (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.

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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

10.6K Views.  Yonsei University.  Wij rapporteren een methode voor het geneesmiddel sorptie evaluatie met behulp van een pomp en model drugs (diazepam en tacrolimus). Na drug analyse met behulp van high-performance vloeistofchromatografie, geneesmiddelconcentraties en sorptie niveaus in de buizen van toedieningssystemen worden berekend. 

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

A Cost-effective and Reliable Method to Predict Mechanical Stress in Single-use and Standard Pumps

Pumps are mainly used when transferring sterile culture broths in biopharmaceutical and biotechnological production processes. However, during the pumping process shear forces occur which can lead to qualitative and/or quantitative product loss. To calculate the mechanical stress with limited experimental expense, an oil-water emulsion system was used, whose suitability was demonstrated for drop size detections in bioreactors1. As drop breakup of the oil-water emulsion system is a function of mechanical stress, drop sizes need to be counted over the experimental time of shear stress investigations. In previous studies, the inline endoscopy has been shown to be an accurate and reliable measurement technique for drop size detections in liquid/liquid dispersions. The aim of this protocol is to show the suitability of the inline endoscopy technique for drop size measurements in pumping processes. In order to express the drop size, the Sauter mean diameter d32 was used as the representative diameter of drops in the oil-water emulsion. The results showed low variation in the Sauter mean diameters, which were quantified by standard deviations of below 15%, indicating the reliability of the measurement technique.

Shear stress investigations on an oil-water emulsion system result in drop breakup over the experimental time. To count drop sizes in pumping processes, the suitability of inline endoscopy was successfully demonstrated in this protocol.

Pumps are mainly used when transferring sterile culture broths in biopharmaceutical and biotechnological production processes. However, during the pumping ...

Engineering

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.  (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

Biology

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

Environment

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Bioengineering

In Situ Simulated Anaerobic Method for Culturing Environmental Microbes

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

Author Spotlight: Unraveling the Mysteries of Terrestrial Anaerobic Microorganisms in Uncharted Environments by In Situ Culturing

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

A Protocol to Set Up Needle-Free Connector with Positive Displacement on Central Venous Catheter in Intensive Care Unit

Needle-free connectors were initially designed and promoted to avoid blood exposure for healthcare workers. Some recent data suggest that the latest generation of connectors (with positive displacement) may be of interest for reducing central venous line infections. We have been using needle-free connectors for several years in our intensive care unit and here we present a protocol for installing these connectors on central venous catheters. After insertion of the catheter and control of the permeability of the lines, the connectors must be purged with 0.9% NaCl before being connected. The connectors replace all disposable caps used on infusion stopcocks and manifolds. All the connectors are changed every 7 days as recommended by the manufacturer (except when there is macroscopic contamination, which requires an immediate change of the connector). Before each injection, the connector must be disinfected for at least 3 seconds with 70% isopropyl alcohol. The connectors must not be disconnected (unless changed), as the injection is done through the device. Setting up the connectors slightly increases the total time required to place the catheter and there is no formal evidence that these connectors reduce the incidence of infectious or thrombotic complications. However, these devices simplify the management of central venous lines and prevent the catheter circuit from &#34;opening&#34; once it has been sterilely installed.

We present a protocol to show the installation of a needle-free connector with positive displacement on a central venous catheter.

Needle-free connectors were initially designed and promoted to avoid blood exposure for healthcare workers. Some recent data suggest that the latest ...

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 &#176;C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Presented here is a protocol for a standardized in vitro hemodynamic loop model. This model allows to test the hemocompatibility of perfusion tubes or vascular stents to be in accordance with ISO (International Organization for Standardization) standard 10993-4.

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive ...

Localized Delivery of GLP-2 via PVA-Coated Medical Devices for Therapeutic Use

Validation of Therapeutic Agent Conjugation to Polyvinyl Alcohol-Coated Medical Devices

Protein-based therapeutics are often limited by their route of administration and inability to confine them to their site of action. One innovative approach we have developed is to covalently bind protein therapeutics to medical devices, allowing more localized and highly concentrated delivery of these agents to their intended site of action. This study aims to evaluate if glucagon-like peptide-2 (GLP-2) can be covalently bound to the vaginal expansion sleeve (VES) and intestinal expansion sleeve (IES) devices in clinically relevant and measurable quantities.
Expansion sleeves were coated with polyvinyl alcohol (PVA) and crosslinked with glutaraldehyde/sulfuric acid vapor to create a chemically active surface capable of binding amine-containing therapeutics such as GLP-2. A standard curve was created by binding 250 &#181;g, 100 &#181;g, 50 &#181;g, 25 &#181;g, and 0 &#181;g of GLP-2 to PVA-coated wells of a 24-well plate. An ELISA standard was created using a rabbit anti-GLP-2 antibody followed by a goat anti-rabbit IgG alkaline phosphatase secondary antibody plus an alkaline phosphatase blue microwell substrate. Colorimetry (yellow to blue at 620 nm) was proportional to the concentration of GLP-2 bound antibodies, enabling calculation of the bound concentration of GLP-2 on the PVA-coated sleeves. The addition of 50 &#181;g of GLP-2 to IES/VES devices bound an average of 22.69 &#177; 9.32 &#181;g/cm2 of GLP-2 with an external IES/VES surface area (9.425 cm2), indicating that 44% of added GLP-2 was immobilized on the PVA coated IES/VES sleeves.
Current human GLP-2 dosing is 50 &#181;g/Kg. Because each sleeve carries 22 &#181;g, this is approximately 44% of a systemic dose in a single device. This methodology makes it possible to add dramatically lower doses of therapeutic agents to get the same effect as systemic administration of the GLP-2 drug while also avoiding systemic effects.

Here, we present a protocol for derivatizing medical devices with protein therapeutics by conjugating protein therapeutics to a polyvinyl alcohol membrane casted on medical devices. Conjugation of protein-based therapeutics allows for localized and concentrated delivery and is a cost-effective approach that requires vastly lower drug amounts for dosing while minimizing off-target effects.

Protein-based therapeutics are often limited by their route of administration and inability to confine them to their site of action. One innovative ...

A Gut-on-a-Chip Model to Study the Gut Microbiome-Nervous System Axis

The human body is colonized by at least the same number of microbial cells as it is composed of human cells, and most of these microorganisms are located in the gut. Though the interplay between the gut microbiome and the host has been extensively studied, how the gut microbiome interacts with the enteric nervous system remains largely unknown. To date, a physiologically representative in vitro model to study gut microbiome-nervous system interactions does not exist. 
To fill this gap, we further developed the human-microbial crosstalk (HuMiX) gut-on-chip model by introducing induced pluripotent stem cell-derived enteric neurons into the device. The resulting model, 'neuroHuMiX', allows for the co-culture of bacterial, epithelial, and neuronal cells across microfluidic channels, separated by semi-permeable membranes. Despite separation of the different cell types, the cells can communicate with each other through soluble factors, simultaneously providing an opportunity to study each cell type separately. This setup allows for first insights into how the gut microbiome affects the enteric neuronal cells. This is a critical first step in studying and understanding the human gut microbiome-nervous system axis.

neuroHuMiX is an advanced gut-on-a-chip model to study the interactions of bacterial, epithelial, and neuronal cells under proximal and representative co-culture conditions. This model allows unravelling of the molecular mechanisms underlying the communication between the gut microbiome and the nervous system.

The human body is colonized by at least the same number of microbial cells as it is composed of human cells, and most of these microorganisms are located ...

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

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