Polyvinyl-chloride (PVC) is used in a variety of medical products for its excellent physical characteristics such as flexibility,strength,and transparency. The pure PVC is a relatively rigid and brittle polymer,so it has to be modified by adding plasticizers in order to establish the desired mechanical properties of the material. Di(2-ethylhexyl) phthalate (DEHP) is the most commonly used plasticizer for PVC medical devices. However,the chemical substance is able to migrate from the polymer into the contact media and might lead to an unwanted exposure of the patients. There are many reports concerning the release of DEHP from the contact materials [1, 2, 3, 4, 5, 6, 7, 8].
The previous studies have shown that DEHP exhibits reproductive,developmental and testicular toxicities [9, 10, 11]. Based on these findings,the European Directive 2007/47/CE has restricted the use of DEHP in medical devices [12]. At present,several different plasticizers are widely used in the medical devices,such as tris(2-ethylhexyl) trimellitate (TOTM),di-isononyl-cyclohexane-1,2-dicarboxylate (DINCH),acetyl tributyl citrate (ATBC) and di(2-ethylhexyl) terephthalate (DEHT). Bernard et al. [13] recently published a review that outlined the characteristics and toxicities of the plasticizers used currently in PVC medical devices,and the recent analytical methods for determination and quantification of several plasticizers in some matrices. TOTM,an ester of trimellitic acid,is commonly used as an alternative plasticizer in medical devices. It has been reported that TOTM showed weaker hepatotoxicity than DEHP [14, 15]. The current investigation on TOTM has shown that this alternative plasticizer can be more strongly binded to PVC than DEHP,so the leaching is reduced [16, 17, 18, 19]. As for their use in medical devices,some therapeutic acts such as artificial nutrition,transfusion,extracorporeal membrane oxygenation and intravenous lipophilic drug infusion,are considered at risk of exposing patients to plasticizers,due to the plasticizers can be leaded in such clinical situations and can have a negative effect on their health. Kambia et al. [3] have shown that less TOTM and DEHP were apparently released from haemodialysis tubing plasticized with TOTM-DEHP than with DEHP only. Ito et al. [16] have studied the TOTM released from PVC medical devices into Sandimmun ,Prograf and Florid -F using liquid chromatography-tandem mass spectrometry (LC-MS/MS). But for other pharmaceutical solutions,there are few reports in the literature concerning the release behaviors of DEHP and TOTM.
The aim of this study is to describe a validated analytical method (in a single HPLC-UV analysis) to quantify the cumulative amounts of the DEHP and TOTM released from the PVC medical devices into various pharmaceutical solutions,including the lipophilic substances (paclitaxel),parenteral nutrition (fat emulsion injection),acid and alkali pharmaceutical solution (furosemide and levofloxacin hydrochloride injection). We hope that this investigation can provide the essential experimental data for the clinical use of the PVC medical devices.
DEHP (99.7% ) and TOTM (99% ) were purchased from Sigma-Aldrich. Their chemical structures and CAS registry numbers are shown in Fig.1. HPLC-grade methanol was purchased from Merck (Germany). Ultra-pure water was prepared by Milli-Q Gradient ultra-pure water system (Millipore) and used throughout the experiments. All of other reagents used were analytical grade.
Glassware was mainly used to avoid contamination by the plasticizers in plastic consumables. It was systematically washed after each use as previously described by Genay et al. [20]. Some plastic consumables were systematically checked before analysis to be plasticizer-free.
The PVC infusion sets as test materials (length: 1 600 mm,inside diameter: 2.0 mm,thickness: 0.6 mm),which plasticized with DEHP or TOTM separately,were kindly supplied by the manufacturer. The DEHP or TOTM contents of the PVC infusion sets were 35% (m/m).
The intravenous preparations used for the DEHP and TOTM released test were paclitaxel injection (Shenzhen Main Luck Pharmaceuticals Inc.,China),fat emulsion injection (Sichuan Kelun Pharmaceutical Co.,LTD.,China),furosemide (Shanghai Harvest Pharmaceutical Co.,LTD.,China),and levofloxacin hydrochloride injection (Yangtze River Pharmaceutical Group,China). The above injections were purchased from commercial companies (Table 1). Physiological saline (0.9% (m/v) sodium chloride solutions) was also purchased from Sichuan Kelun Pharmaceutical Co.,LTD.,China.
A stock solution of DEHP or TOTM was prepared in methanol at 2 mg/mL in a volumetric flask and stored at 4 ℃. The working solutions for sample spiking and the calibration curves of DEHP and TOTM were diluted with methanol to mass concentrations ranging from 200 to 0.025 μ g/mL and from 80 to 0.04 μ g/mL,respectively. The mass concentration of DEHP or TOTM in the samples was obtained by extrapolation from this calibration plot.
The paclitaxel,furosemide and the levofloxacin hydrochloride solutions were prepared with the 0.9% (m/v) sodium chloride solutions according to the highest concentration in clinical use (Table 1). The fat emulsion injection was directly used without any treatment. The above injections were stored in glass vials before use,and pumped through the investigated infusion set tubes at a flow rate of 1.0 mL/min at 37 ℃ for 24 h. All the injections were studied at 0,2,4,6,8,12,24 h during the experiment.
Chromatographic analysis was performed using a Shimadzu 20A HPLC system equipped with a constant flow-rate pump,an automatic sampling system and a constant-wavelength ultraviolet light detector operated at 274 nm and 230 nm for DEHP and TOTM respectively. The separation was achieved using a 5 μ m SPOLAR C18 column (4.6 mm i. d.×250 mm length) for DEHP and a 5 μ m SPOLAR C8 column (4.6 mm i. d.×250 mm length) for TOTM,and the column oven temperature was maintained at 37 ℃. The flow rate was 1.0 mL/min and the injection volume was 20 μ L. The mobile phase was a mixture of methanol and purified water. Under the optimized conditions,the mobile phase was adjusted and summarized in Table 2 and 3. The mobile phase was filtered through a 0.45 μ m membrane and degassed under a helium stream before use.
The paclitaxel injection,which has passed the PVC infusion sets,was directly analyzed without further preparation. For the furosemide and levofloxacin hydrochloride solutions,10 mL solution was dried and then dissolved with 10 mL methanol before HPLC analysis. The fat emulsion solution needed an extraction procedure before chromatographic analysis. The fat emulsion solution was extracted according to the previously published method [4]. Each fat emulsion sample (100 mL) was subjected to extraction with n-hexane (100 mL). The mixture was extracted and the separated organic layer (fraction 1) was transferred to a clean conical glass tube. The aqueous phase was extracted again with 200 mL n-hexane,and the mixture was treated as above. The separated organic phase (fraction 2) was combined with fraction 1,and the total organic phase was evaporated to dryness in a thermostatic oven at 40 ℃. The residue was dissolved in 10 mL ethanol. After centrifugation,20 μ L of supernatant was finally injected into the chromatograph.
After the optimization of HPLC conditions,the method was validated using the DEHP or TOTM standard solution. The quantification was achieved by external calibration using standard solution of DEHP or TOTM according to the standard addition method. The linear regression was performed using the ratio of DEHP or TOTM peak area plotted against the concentration.
The method was applied to the pharmaceutical solutions spiked with 0.5,10,80 μ g/mL DEHP or TOTM for the recovery test. All the samples were determined by triplicate analyses. In order to ascertain the chemical stability of the plasticizer solutions,a stability study was performed for the validation standard solutions at different times: 1,2,4,6,8,12,24 and 48 h.
The concentrations of DEHP and TOTM from the PVC infusion sets were determined in triplicate for each pharmaceutical solution. And the cumulative amount of DEHP (or TOTM) was obtained by multiplied the mean concentration of DEHP (or TOTM) by the volume of pharmaceutical solution.
Moreover,in order to ascertain the chemical stability of the plasticizer solutions,a stability study was performed at room temperature and ambient light with the validation standard solutions for 0,1,2,3,4,10 and 15 d.
An analytical method for the quantification of DEHP or TOTM in PVC products was developed and published [3]. In order to adapt this method to the quantification of DEHP or TOTM in PVC infusion sets from various pharmaceutical solutions,a mixture of ethanol/water was selected as the mobile phase. And the ratio of ethanol to water was adjusted to make sure that there was no peak interference from other compounds in the extraction solvents. The resulting chromatograms with the elution conditions are presented in Fig. 2 and Fig. 3. The retention times are presented in Table 2 and Table 3.
Just as shown in the Fig. 2 and Table 2,the retention times of DEHP were approximately 10.4 min and 24.3 min,when the volume ratio of methanol to purified water was adjusted from 95 ∶ 5 to 90 ∶ 10,respectively. Compared with the chromatogram of DEHP standard solution,there was no other interference peaks displayed in the chromatograms.
The limit of detection (LOD,S/N≥2) and the limit of quantification (LOQ,S/N≥7) of DEHP are shown in Table 2. For DEHP measurement,the calibration curve was obtained by plotting the DEHP peak areas versus their concentrations,and one calibration curve was obtained for each pharmaceutical solution. They were linear over the range of 0.025-200 μ g/mL for fat emulsion,furosemide and levofloxacin hydrochloride solutions and 0.11-200 μ g/mL for paclitaxel solution. The linear regression equations obtained were y=3 650.9 857x-2 834.954 (r=0.999 9) for fat emulsion,furosemide and levofloxacin hydrochloride solutions,and y=3 342.4 888x-2 306.7 174 (r=0.999 8) for paclitaxel solution (y,peak area; x,DEHP mass concentration,μ g/mL). Then we examined the recoveries using each intravenous preparation. The average recoveries of DEHP in fat emulsion,furosemide,levofloxacin hydrochloride and paclitaxel solutions were 97.42% -102.64% (RSD=1.03% ),100.34% -108.35% (RSD=1.07% ),96.68% -104.30% (RSD=1.33% ) and 94.75% -101.77% (RSD=1.34% ),respectively. Recoveries between 80% and 120% were considered as acceptable regarding sample preparation and trace level analysis. These results indicate that there was no significant loss of samples during the preparation step.
The results of the developed method for TOTM are shown in Fig. 3 and Table 3. It can be seen that the retention times of TOTM were 15.489 min and 15.387 min in the furosemide solution and levofloxacin hydrochloride solutions respectively,while the volume ratio of methanol to water was 94 ∶ 6 in the mobile phase. When the volume ratio of methanol to water was adjusted to 92 ∶ 8,the retention time of TOTM in the fat emulsion solution was 18.654 min. When the volume ratio was 90 ∶ 10,the retention time of TOTM in the paclitaxel solution was 31.486 min. The peaks of TOTM in all the extracts were free from the interference of impurity peaks.
With the exception of paclitaxel solution,the LOD and LOQ of TOTM in the other test solutions were 12.5 ng/mL and 25 ng/mL respectively. While for the paclitaxel solution,the LOD and LOQ were 48.0 ng/mL and 110 ng/mL respectively. In the furosemide and levofloxacin hydrochloride solution,the TOTM linear equation was y=24 087.34x-2 384.57 (r=1.000 0); in the fat emulsion extract,the TOTM linear equation was y=24 349.40x-3 633.93 (r=1.000 0); in the paclitaxel solution,the TOTM linear equation was y=28 888.07x-4 990.34 (r=0.999 6) (y,peak area; x,TOTM mass concentration,μ g/mL). In the four pharmaceutical solutions,the TOTM recoveries were 93% -105% ,indicating that the present detection method is reliable and steady.
The intra-day precision or repeatability,which expresses random error,was less than 0.8% . The inter-day precision or intermediate precision was less than 3% . None of the analyses exceeded 5% of the initial value of the DEHP and TOTM solutions. The solutions are therefore considered stable for 48 h.
The average recoveries of DEHP and TOTM in the fat emulsion pharmaceutical solution were 95% -102% (Table 4). Based on these result,this extraction technique can be adapted to the extraction of DEHP or TOTM from the fat emulsion solution.
The proposed method was applied to the determination of DEHP and TOTM released from the PVC infusion sets in various pharmaceutical solutions. Since the cumulative amount of plasticizers increases with the rise of temperature,we set the extraction temperature at 37 ℃ to simulate the highest temperature in the clinic application. Besides,the treatment time for these disposable infusion products only lasted for 24 h,thus we set the detection time as 24 h in the test. Fig. 4 and Fig. 5 show the cumulative amounts of DEHP and TOTM time-course obtained during a 24-hour leaching experiment.
For the PVC infusion products with DEHP as the plasticizer,detectable DEHP was found both in the paclitaxel and fat emulsion pharmaceutical solutions at the beginning of the treatment,and the cumulative amounts of DEHP released increased with the increase of treatment time. In the paclitaxel solution (the extraction time was less than 12 h) and the fat emulsion solution,the cumulative amounts of DEHP released increased linearly. This observation is consistent with the fact that the plasticizer might accumulate in the leaching solutions,due to lipophilic characteristics. While in the furosemide and levofloxacin hydrochloride solutions,no released DEHP was detected within the first two hours of the experiment. During the whole test,the cumulative amounts of DEHP in the furosemide and levofloxacin hydrochloride solutions slightly increased,and the amounts of released DEHP in these two solutions were far less than that in the fat emulsion and paclitaxel solution. After extracted for 24 h,the cumulative amounts of DEHP released from the PVC infusion set in paclitaxel solution,fat emulsion solution,levofloxacin hydrochloride and furosemide solutions were 21.14 mg,0.38 mg,0.075 mg and 0.035 mg,respectively. It can be seen that the cumulative amount of DEHP in paclitaxel solution was 55 times of that in fat emulsion solution,more than 200 times of that in levofloxacin hydrochloride solution and more than 600 times of that in Furosemide solution.
For the disposable infusion products with TOTM as the plasticizer,the cumulative amount of TOTM is shown in Fig. 5. The detectable concentrations of TOTM were not found in the levofloxacin hydrochloride and furosemide solutions at the beginning of the treatment. After 6 h and 12 h treatments,certain amounts of TOTM were found in the levofloxacin hydrochloride and furosemide solution. In the paclitaxel and fat emulsion solutions,detectable TOTM were found even at the initial stage of the treatment,and the cumulative amounts of TOTM were in linear growth with the increase of time. After extracted for 24 h,the cumulative amounts of TOTM released from the PVC infusion set in paclitaxel,fat emulsion,levofloxacin hydrochloride and furosemide solutions were 0.078 mg,0.067 mg,0.025 mg and 0.011 mg,respectively. It can be seen that the cumulative amount of TOTM was far less than that of the released DEHP. The amount of released DEHP was 271 times of that of the released TOTM. The results were consistent with the earlier reports [17, 20] suggesting that the TOTM are more strongly bound to the PVC than DEHP,so their leaching is reduced.
In this study,we developed an analytical method using HPLC-UV for the determination of DEHP (or TOTM) released from PVC medical devices. Our results demonstrate that TOTM could be a superior alternative to DEHP for use in medical devices because of its lower leach ability.
2015, Vol. 33 Issue (5): 522-529
PDF (852 KB)
2015, Vol. 33 Issue (5): 522-529
PDF (852 KB)