Applied Radiation and Isotopes 86 (2014) 57–62
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Evaluation of alternative rapid thin layer chromatography systems for quality control of technetium-99m radiopharmaceuticals Kennedy Mang0 era a,b,c,n, Derek Wong a,c, David Douglas b, Kellie Franz a, Taddese Biru a a
Radiopharmacy, Health Sciences Centre, GC219-820 Sherbrook Street, Winnipeg, MB, Canada R3A1R9, Radiology, Faculty of Medicine, University of Manitoba, GA216-820 Sherbrook Street, Winnipeg, MB, Canada R3T 2N2 c Faculty of Pharmacy, University of Manitoba, 750 McDermot Avenue, Winnipeg, MB, Canada R3E 0T5 b
H I G H L I G H T S
Whatman 3MM™ paper and Tec-Control™ systems validated as ITLC-SG alternatives. Full solvent migration in Tec-Control™ was not reproduced for 99mTc-tetrofosmin. Systems are comparable to ITLC-SG, Tec-Control™ is more conservative for 99mTc-sestamibi. Tec-Control™ ( 1 min) and Whatman 3MM™ (7–9 min) are faster than ITLC-SG (10–15 min). Overall, Tec-Control™ is preferred but Whatman 3MM™ recommended for 99mTc-tetrofosmin.
art ic l e i nf o
a b s t r a c t
Article history: Received 16 September 2012 Received in revised form 1 October 2013 Accepted 16 December 2013 Available online 8 January 2014
Whatman 3MM™ and Tec-Control™ systems were evaluated as ITLC-SG alternatives for 99mTcradiopharmaceuticals. They compare well in accuracy and reproducibility, and are faster and more convenient than ITLC-SG. Tec-Control™ radiochemical purity values for 99mTc-sestamibi were more conservative than ITLC-SG. Full solvent migration was not reproduced for 99mTc-tetrofosmin in TecControl™, and for this Whatman 3MM™ is preferred. Developing times were 10–15 min, 7–9 min and 1 min for ITLC-SG, Whatman 3MM™ and Tec-Control™, respectively. Overall, Tec-Control™ strips are preferred due to speed and ease of use. & 2013 Elsevier Ltd. All rights reserved.
Keywords: Quality control ITLC-SG Whatman 3MM™ Tec-Control™ Technetium Radiopharmaceuticals
1. Introduction Silica gel-impregnated instant thin layer chromatography (ITLC-SG) strips have been in widespread use in the quality control of radiopharmaceuticals for over three decades (Saha, 2010). The major advantages of a validated ITLC analytical process have been the speed of the test and its relative simplicity and inexpense. The primary vendor of ITLC-SG strips (Pall Life Sciences, Ann Arbor, MI) discontinued supply in 2008, with severe implications for everyday radiopharmacy practice, eased later by availability of the product from a different vendor (Agilent, product SGI0001). This discontinuation followed that of polysilicic acid-impregnated ITLC n Corresponding author at: Radiopharmacy, Health Sciences Centre, GC219-820 Sherbrook Street, Winnipeg, MB, Canada R3A1R9. Tel.: þ 1 204 7873540; fax: þ1 204 787 1313. E-mail address: [email protected] (K. Mang0 era).
0969-8043/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.apradiso.2013.12.016
strips in 2004/5, which were widely used for the quality control of 99mTc-dimercaptosuccinic acid and other 99mTc-iminodiacetic acid (IDA) complexes. Subsequently, an alternative solid extraction procedure for the 99mTc-mebrofenin was developed and validated, applicable to other 99mTc-IDAs (Billinghurst et al., 2004). A number of groups have also reported on alternative quality control systems to replace ITLC-SG systems, although most of the literature is applicable to single radiopharmaceutical products (Amin et al., 2011; Eggert et al., 2010; Ponto, 2011; Zlata et al., 2010). The central radiopharmacy at Winnipeg Health Sciences Centre supplies and provides services to five hospitals affiliated to the Winnipeg Regional Health Authority and prepares a variety of radiopharmaceuticals. QC operations would therefore have been severely impacted by the discontinuation of the commercial ITLCSG strips and there was strong motivation to identify and validate QC tests alternative to the routine ITLC-SG systems (Table 1),
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Table 1 Routine chromatography systems used at the radiopharmacy. Product
Media
Manufacturer and model
Solvent
99mTc-DTPA
ITLC-SGa,b
Pall Corp. 61886
99mTc-MDP
ITLC-SGa,b
Pall Corp. 61886
Methylethyl ketone 0.9% Saline Methylethyl ketone 0.9% Saline 0.9% Saline Ethanol Methylethyl ketone Methylethyl ketone Ethyl Acetate
a
99mTc-SbS colloid 99mTc-Sestamibi 99mTc-DMSA 99mTc-Sulfur colloid 99mTc-Tetrofosmin a b c
ITLC-SG Aluminum Oxide IB-Fc ITLC-SGa ITLC-SGa ITLC-SGa
Pall Corp. 61886 J.T. Baker 4467-02 Pall Corp. 61886 Pall Corp. 61886 Pall Corp. 61886
Developed in a 22 6 cm glass cylindrical tank. Developed in a 30 30 cm rectangular chamber. Developed in a 18 12 cm rectangular chamber.
Table 2 ITLC chromatography systems based on Tec-Control™ miniaturized strips. Product
Media
Strips color-codes
Solvent
Developing vial (mL)
99mTc-DTPA
Whatman 31 ET ITLC-SG
Red
Acetone
10
Black
Distilled water Acetone
99mTc-MDP
Whatman 31ET ITLC-SG
99mTc-SbS colloid 99mTc-Sestamibi
ITLC-SG Whatman 31ET 99mTc-DMSA Whatman 31ET 99mTc-Sulfur colloid Whatman 31ET 99mTc-Tetrofosmin Whatman 1
Red Black
Product
Solvent
99mTc-MDP
Methylethyl ketone 0.9% Saline Methylethyl ketone 0.9% Saline Ethyl Acetate Acetone: Dichloromethane
99mTc-SbS colloid 99mTc-Sulfur colloid 99mTc-Tetrofosmin 10
10 5
Yellow
Distilled water 0.9% Saline Ethyl acetate Acetone
Red
Acetone
10
Teal
Ethyl acetate
5
Dark green Pink
Table 3 Chromatography systems based on Whatman 3MM™ strips.
10
technetium radiolabelled complex after subtraction of tested radiochemical impurities (free pertechnetate, reduced hydrolyzed technetium). Preparation and evaluation of the effect of vial size and shape on RCP following the Tec-Control procedures was determined using two vial sizes, 5 mL (Biodex, Shirley, NY) and 10 mL (Greer Laboratories, Lenoir, NC). 2.2. Quality control procedures
including the commercial options Tec-Control™ chromatography (Biodex, Shirley, NY; Table 2) (Barnes et al., 1996; Zimmer, 2004) and Whatman 3MM™ paper system (Table 3). Tec-Control systems are available in color-coded strips for specific radiopharmaceuticals. These systems were compared with the standard ITLC-SG procedures routinely applied at our radiopharmacy.
2. Materials and methods 99mTc-sodium pertechnetate was eluted from Tecknekow™ generators (Mallinckrodt, St. Louis, MO). 2.1. Preparation of 99mTc-radiopharmaceuticals Commercial kits of diethylene triaminopentaacetic acid (DTPA, DraxImage), methylene phosphonate (MDP, DraxImage), sestamibi (Covidien and Cardiolite, Lantheus Medical), dimercaptosuccinic acid (DMSA, GE Healthcare) and sulfur colloid (GE Healthcare) were reconstituted according to manufacturer product inserts to make 99mTc-radiopharmaceuticals. Procedures for preparation of 99mTc-tetrofosmin (Myoview, GE Healthcare) were adopted from the manufacturer0 s product insert, but higher activities (up to 25 GBq 99mTc-pertechnetate) were used in reconstitution. 99mTcantimony sulphide (99mTc-SbS) colloid was prepared using inhouse kit formulations and following established procedures (Billinghurst and Jette, 1979). For all preparations, the pass limits for radiochemical purity is 90%, calculated as the net percentage
All spotting of product samples onto the strips were performed using 28 G, 1 cm3 insulin syringes (VanishpointTM, Retractable Inc., Little Elm, Texas). Various operators were involved in conducting the QC tests, but in all cases the same person performed each comparative test set. For detection of radiochemical species, the chromatography strips were run on an in-house TLC scanner equipped with a 5 cm NaI(Tl) detector and connected to an Eagle Plus MCA system and a Genie-200 Basic software (Canberra, Concord, Ontario). The comparative time delay to full development of the spotted strips by the relevant solvent in the chamber was determined for the two alternative strip systems and compared to the development times of the routine ITLC-SG systems. (a) Routine ITLC-SG and thin layer chromatography (TLC) Table 1 summarizes the routine ITLC-SG chromatography systems for each product. The strips (1.5 20 cm) were cut from 20 20 cm impregnated glass fiber sheets (Pall Life Sciences, Ann Arbor, MI). Product was spotted 2.5 cm from the bottom and developed in a closed 22 6 cm glass cylindrical tank or a closed 30 30 cm developing chamber, which contained the appropriate developing solvent. When solvent migrated to a mark 2.5 cm from the top, the strips were immediately removed, left to dry (except where solvent was saline), and then wrapped in plastic film and analyzed on the TLC counter. 99mTc-sestamibi analysis requires use of aluminum oxide IB-F chromatography strips (J.T. Baker, Phillipsburg, NJ, USA). The pre-cut 2.5 7.5 cm strips were solvated with a drop of
K. Mang0 era et al. / Applied Radiation and Isotopes 86 (2014) 57–62
ethanol, spotted with the product and air-dried before developing in ethanol in an enclosed 18 12 cm rectangular developing chamber. The strips were removed when the solvent front was 1 cm from the top, wrapped in plastic film and analyzed on the TLC counter. (b) Tec-Control™ chromatography The Tec-Control™ chromatography systems evaluated for the various radiopharmaceuticals are summarized in Table 2. The procedures using Tec-Control™ chromatography strips were adopted from literature supplied by the vendor (Zimmer, 2004). The color-coded miniaturized strips are supplied pre-cut as 6 0.7 cm strips (7 0.7 cm for 99mTc-sestamibi). A small drop of product was spotted on the marked origin line (1 cm from the bottom) and the strips developed in their appropriate solvents in either “off-the-shelf” 10 mL vials (Greer Laboratories, Lenoir, NC), or the suggested 10 mL and 5 mL vials (Biodex, Shirley, NY). Once the strips were developed to the marked solvent front line (1 cm from the top), they were immediately removed, wrapped in plastic film and analyzed on the TLC counter. (c) Whatman 3MM™ paper Table 3 summarizes the chromatography systems based on Whatman 3MM™ paper that were evaluated for various radiopharmaceuticals. Whatman 3MM™ paper (Whatman Inc., Clifton, NJ) was obtained as 20 20 cm sheets, and cut into 1.5 7.5 cm strips. Evaluating these strips involved spotting the product 1 cm from the bottom and allowing them to develop to a mark 1 cm from the top. The strips were developed in an enclosed 18 12 cm rectangular chamber, filled with the appropriate solvent. The strips were immediately removed when solvent front was reached, wrapped in plastic film and analyzed on the TLC counter. Chromatography strips developed in acetone:dichloromethane were air-dried before being wrapped in plastic film and analyzed on the TLC counter.
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Table 4 Radiochemical purity values (%) obtained for 99mTc-DTPA preparations analyzed using routine ITLC-SG and miniaturized Tec-Control™ systems. 99mTc-DTPA—Routine system versus miniaturized system (N ¼7) Routine system
Range RCP
Median RCP
Average RCP
7 STD
ITLC-SG/MEK ITLC-SG/saline Aggregate RCP
99.6–99.9 99.3–99.9 98.9–99.8
99.9 99.8 99.7
99.8 99.7 99.5
0.12 0.25 0.37
Miniaturized system
Range RCP
Median RCP
Average RCP
7 STD
Tec-Control™/acetone Tec-Control™/water Aggregate RCP
98.4–99.9 98.4–99.7 96.5–99.6
99.3 99.3 98.6
99.3 99.0 98.3
0.51 0.75 1.06
Table 5 Radiochemical purity values (%) obtained for 99mTc-MDP preparations analyzed using routine ITLC-SG and miniaturized Tec-Control™ systems (A), and radiochemical purity values (%) obtained for 99mTc-MDP preparations analyzed using routine ITLC-SG and Whatman 3MM™ paper strips (B). (A) 99mTc-MDP—Routine system versus miniaturized system (N ¼ 17) Routine system
Range RCP
Median RCP
Average RCP
7STD
ITLC-SG/MEK ITLC-SG/saline Aggregate RCP
95.6–99.7 95.8–99.6 91.4–98.9
98.8 99.2 98.2
98.4 98.4 96.8
1.35 1.35 2.42
Miniaturized system
Range RCP
Median RCP
Average RCP
7STD
Tec-Control™/acetone Tec-Control™/water Aggregate RCP
88.2–99.9 96.5–99.8 87.9–99.7
99.7 99.7 99.4
98.8 99.5 98.3
2.79 0.83 2.82
(B) 99mTc-MDP—Routine system versus 3MM™ paper system (N ¼19) Routine system
Range RCP
Median RCP
Average RCP
7STD
ITLC-SG/MEK ITLC-SG/saline Aggregate RCP
96.2–99.8 85.2–99.5 83.9–98.4
98.6 98.7 96.9
98.3 97.8 96.1
0.90 3.20 3.17
3. Results and discussion
3MM™ paper system
Range RCP
Median RCP
Average RCP
7STD
The effectiveness of the miniaturized Tec-Control and the Whatman 3MM chromatography paper strips was studied against routine ITLC-SG QC chromatography methods and using routine 99mTc-kit preparations at the central radiopharmacy. Paired t-test calculations in runs with adequate number of samples are all twotailed. Developing times of each set of strips were monitored. Generally, the routine ITLC-SG strips took the longest to develop at approximately 10 min to 15 min. The Whatman 3MM™ paper strips took 7 min to 9 min to develop, whereas the miniaturized Tec-Control™ strips developed in about a minute. The proposed systems also show largely similar and comparable chromatographic (Rf) profiles to the corresponding ITLC-SG methods.
Tec-Control™/acetone Tec-Control™/water Aggregate RCP
95.7–99.9 91.8–99.1 90.8–98.1
98.8 97.9 95.3
98.4 97.2 95.6
0.95 1.97 2.04
3.1. 99mTc-DTPA Assessing % RCP of 99mTc-DTPA requires a two-strip system to demarcate impurity levels of free pertechnetate and reduced hydrolyzed technetium. The results summarized in Table 4 compare corresponding ITLC-SG and Tec-Control™ single strip procedures and compare the aggregate RCP of 99mTc-DTPA after accounting for both impurities. The routine ITLC-SG strips developed in MEK (demarcating reduced hydrolyzed technetium levels) gave an average value of 99.8%7 0.12 while the corresponding miniaturized strips developed in acetone gave an average value of 99.3% 70.51. Routine
strips developed in 0.9% saline (demarcating free pertechnetate levels) gave average value of 99.7% 70.25 while the corresponding miniaturized strips developed in distilled water gave average value of 99.0% 70.25. The miniaturized strips gave an average RCP for 99mTc-DTPA of 98.3% 7 1.06, a value that is comparable to the aggregate RCP of 99.5% 70.37 determined using routine strips. 3.2. 99mTc-MDP As for 99mTc-DTPA, two-strip systems were used to determine separately the levels of the two expected impurities in 99mTcMDP and an aggregate RCP was calculated for the preparation. Results are presented in Table 5. Routine ITLC-SG strips developed in MEK to demarcate colloid impurity measured a purity average of 98.4% 7 1.35 while the corresponding miniaturized strips developed in acetone measured 98.8% 72.79. For free pertechnetate impurity, routine strips developed in 0.9% saline measured an average RCP of 98.4% 71.35 (note: coincidentally, values are identical to MEK above but data set is different) and the miniaturized strips developed in distilled water measured 99.5% 70.83. Routine strips determined
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an aggregate RCP of 96.8% 7 2.42 while the miniaturized strips gave an average aggregate RCP of 98.3% 72.82. Paired two-tailed t-test results showed that the overall RCP values for 99mTc-MDP preparations are not statistically different for the miniaturized strips compared to the routine ITLC-SG strips used in daily QC procedures (p¼ 0.588). Paradoxically, although the miniaturized system gave a higher average RCP value, for the one sample where there was a quality significance, this system gave an overall RCP of 87.9% (product fails QC), compared with 92.5% (product passes QC) obtained for the ITLC-SG system. The one result appears to be an anomaly. Whatman 3MM™ paper strips were also compared to the routine strips (Table 5B). The routine strips developed in organic solvent detected a RCP of 98.3 70.90%, whereas Whatman 3MM™ paper strips had an average % RCP of 98.4% 70.93. The results of both sets of strips correlated well with each other. The routine strips developed in saline detected a RCP of 97.8% 73.20, whereas the Whatman 3MM™ paper strips gave 97.2% 71.97. Again, the % RCP results correlated well with each other. Millar et al. (2010) evaluated five Whatman papers (1CHR, 31ET, 3MM, 4CHR and 54SFC) using methyl ethyl ketone for detecting 99mTc-pertechnetate and using sodium acetate solution (136 g/L) for detecting reduced hydrolysed technetium impurities. They found the highest resolutions with 1CHR, 4CHR and 54SFC and erroneously high levels of colloidal and 99mTc-pertechnetate when using 1CHR and 4CHR. They proposed 54SFC paper as a suitable alternative to ITLC-SG for measuring radiochemical impurities in 99mTc-MDP preparations. Paired two-tailed t-test for our results showed that the overall RCP values for 99mTc-MDP preparations are statistically significantly lower for the Whatman 3MM™ strips compared to the routine ITLC-SG strips used in daily QC procedures (p¼ 0.025). The significance of results of paired t-test values for the individual systems delineating the impurities free pertechnetate (saline is solvent) and RHT colloid (organic solvent) were mixed, with values of p ¼0.014 and p ¼0.708, respectively. The somewhat generally lower overall RCP values obtained for the miniaturized system had a practical quality significance in one sample where an overall RCP of 87.9% was obtained, compared with 92.5% obtained for the ITLCSG system. For this sample, the ‘fail0 and ‘pass’ conclusions for quality are therefore conflicting, and the miniaturized system appears to provide more conservative results for quality when compared to ITLC-SG analysis.
3.3. 99mTc-Antimony sulphide (99mTc-SbS) colloid Comparative quality control for 99mTc-antimony sulphide (99mTc-SbS) colloid was performed using all three systems, the routine ITLC-SG, the miniaturized Tec-Control™, and the Whatman 3MM™ paper strips. All three systems are a one-strip procedure that distinguishes 99mTc-SbS from free pertechnetate (which migrates to the solvent front). Results comparing routine ITLC-SG and miniaturized Tec-Control™ systems are presented in Table 6A. The routine strips detected an average RCP of 98.8% 70.62 while the miniaturized strips detected a RCP of 98.7% 7 1.60. These values are nearidentical. Results comparing routine ITLC-SG and Whatman 3MM™ paper strips are presented in Table 6B. The routine strips show an average % RCP of 97.6 71.21 Whatman 3MM™ strips give a RCP of 98.0% 7 2.47. Overall, paired t-test analysis shows that the results obtained for the routine and miniaturized strips are similar, with no significant statistical difference (p ¼ 0.872). A similar similarity without statistical significance is seen in the pairing of the routine and Whatman 3MM™ strips samples (p ¼0.611).
Table 6 Radiochemical purity values (%) obtained for 99mTc-SbS Colloid preparations analyzed using routine ITLC-SG and miniaturized Tec-Control™ systems (A), and radiochemical purity values (%) obtained for 99mTc-SbS Colloid preparations analyzed using routine ITLC-SG and Whatman 3MM™ paper strips (B). (A) 99mTc-SbS colloid—Routine system versus miniaturized system (N ¼11) Routine system
Range RCP
Median RCP
ITLC-SG/saline
97.5–99.5
98.6
Miniaturized system
Range RCP
Median RCP
Tec-Control™/saline
95.0–99.9
98.8
Average RCP 7 STD 98.8
0.62
Average RCP 7 STD 98.7
1.60
(B) 99mTc-SbS colloid—Routine system versus 3MM™ paper system N ¼ 13 Routine system
Range RCP
Median RCP
ITLC-SG/saline
95.9–99.5
97.3
3MM™ paper system
Range RCP
Median RCP
3MM/saline
91.0–99.9
98.5
Average RCP 7 STD 97.6
1.21
Average RCP 7 STD 98.0
2.47
Table 7 Radiochemical purity values (%) obtained for 99mTc-Sestamibi preparations analyzed using routine TLC and miniaturized Tec-Control™ systems. 99mTc-Sestamibi—Routine system versus miniaturized system (N ¼ 21) Routine system
Range RCP
Median RCP
Average RCP
7 STD
TLC/ethanol
90.9–98.5
97.9
97.5
1.59
Miniaturized system
Range RCP
Median RCP
Average RCP
7 STD
Tec-Control™/saline
88.1–98.9
97.2
96.7
2.20
3.4. 99mTc-Sestamibi When comparing the routine plates and miniaturized strips for quality control of 99mTc-sestamibi (Table 7, N ¼21), the mean and median RCP obtained using the miniaturized strips (96.7 and 97.2%) are very similar to those obtained by the routine TLC/ ethanol plates (97.5 and 97.9%, respectively). These results obtained from the routine and miniaturized strips were therefore consistent with one another. However, a notable observation for 99mTc-sestamibi results is that for one sample the product passed the limits for quality when measured by the routine procedure (RCP¼90.9%) but failed the limits when determined using the miniaturized strips (RCP¼88.1%). Additionally, paired two-tailed t-test results showed that although the individual paired RCP values were consistently close, they were also consistently highly statistically different (p¼ 0.0005). The RCP values were consistently lower for the miniaturized strips compared to the routine TLC/ethanol system. Although the difference between the two systems is small, the results straddle either side of the pass margin of 90% and this result has clear clinical significance. Overall, the miniaturized system provided more conservative results for quality for 99mTc-sestamibi analysis. In studying a variety of 99mTc-radiopharmaceuticals and comparing ITLC-SG with two alternative procedures (Whatman 3MM™ and Whatman 1MM™) with 99mTc-sestamibi being the common one with our study, Monteiro et al. (2009) obtained near-identical RCP values between the ITLC-SG and the Whatman systems for 99mTc-sestamibi (N¼4, two replicates of two preparations). They monitored the preparations at 30 min, 60 min and 240 min.
K. Mang0 era et al. / Applied Radiation and Isotopes 86 (2014) 57–62
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compare very well to the routine strips, with values of 99.5% 70.38 and 98.6% 71.17, respectively. Results comparing the routine ITLC-SG and Whatman 3MM strips for % RCP are presented in Table 9B. The routine strips detected a RCP of 96.7% 7 0.63, whereas the Whatman 3MM™ paper strips detected a RCP of 97.4% 70.81. Overall, the % RCP values obtained from the Whatman 3MM™ paper strips are consistent with those of the routine ITLC-SG strips.
Paired t-test evaluation of RCP values reported in a similar comparative study by Zimmer, (2004) also show consistently slightly lower values for the miniaturized system compared to the routine TLC system (N ¼13); and that this difference is statistically significant (p¼ 0.033). Their results have mean RCP values for the routine and the miniaturized systems of 97.4% 70.84 and 96.9 71.46, respectively. 3.5. 99mTc-DMSA
3.7. 99mTc-Tetrofosmin
A comparison of corresponding ITLC-SG and Tec-Control™ results obtained for QC of 99mTc-DMSA are summarized in Table 8. Values obtained for RCP are high and virtually identical for the two systems, with average RCP values of 99.5% 70.65 and 99.3% 70.54, respectively.
Our evaluation of 99mTc-Tetrofosmin using the miniaturized chromatography method was unsuccessful as the solvent-phase would only develop two-thirds up the strip (up to the vial’s lip), and would remain there indefinitely. The ethyl acetate eventually evaporated from the vial after approximately fifteen minutes of developing time. Evaluation of Whatman 3MM™ on preparations of 99mTcTetrofosmin was as a two-strip system, and the chromatography strips were developed in both acetone:dichloromethane (DCM) and 0.9% saline in order to separately quantify the impurity levels of free pertechnetate and reduced hydrolyzed technetium, respectively. Results are presented in Table 10. Routine ITLC-SG strips developed in acetone:DCM gave an average aggregate RCP of 96.5% 71.57 whereas Whatman 3MM™ paper strips showed a very similar average aggregate RCP of 96.8% 70.95. Other alternative procedures have been suggested for quality control of 99mTc-tetrofosmin (Hammes et al., 2004). Eggert et al. (2010) evaluated a method adopted from the QC of 99mTc-sestamibi (Hung et al., 1991) that uses solvent saturation pads (Krackeler Scientific, Inc.) as stationary phase and a 1:1 chloroform:tetrahydrofuran mixture as mobile phase. Although they found this alternative method more accurate, they cited a burdensome preparation and refrigeration process for the combination of chloroform and
3.6. 99mTc-Sulfur colloid Results comparing routine ITLC-SG and miniaturized TecControl strips for 99mTc-Sulfur Colloid preparations are presented in Table 9A. The % RCP values given by the miniaturized strips Table 8 Radiochemical purity values (%) obtained for 99mTc-DMSA preparations analyzed using routine TLC and miniaturized Tec-Control™ systems. 99mTc-DMSA—Routine system versus miniaturized system (N ¼ 5) Routine system
Range RCP
Median RCP
ITLC-SG/MEK
98.4–99.9
99.9
Miniaturized system
Range RCP
Median RCP
Tec-Control™/acetone
98.4–99.8
99.5
Average RCP
7STD
99.5
0.65
Average RCP
7STD
99.3
0.54
Table 9 Radiochemical purity values (%) obtained for 99mTc-Sulphur Colloid preparations analyzed using routine TLC and miniaturized Tec-Control™ systems (A), and radiochemical purity values (%) obtained for 99mTc-Sulphur Colloid preparations analyzed using routine ITLC-SG and Whatman 3MM™ Paper System (B). (A) 99mTc-Sulphur colloid—Routine system versus miniaturized system (N ¼ 7) Routine system
Range RCP
Median RCP
Average RCP
7 STD
ITLC-SG/MEK Miniaturized system Tec-Control™/saline
96.7–99.5 Range RCP 96.5–99.6
98.2 Median RCP 99.0
98.6 Average RCP 99.5
1.17 7 STD 0.38
(B) 99mTc-Sulphur colloid—Routine system versus 3MM™ paper system (N¼ 12) Routine system
Range RCP
Median RCP
Average RCP
7 STD
ITLC-SG/MEK 3MM system 3MM/saline
95.9–98.1 Range RCP 96.1–99.3
96.8 Median RCP 97.4/97.5
96.7 Average RCP 97.4
0.63 7 STD 0.81
Table 10 Radiochemical purity values (%) obtained for 99mTc-tetrofosmin preparations analyzed using routine ITLC-SG and Whatman 3MM™ paper strips. 99mTc-Tetrofosmin—Routine system versus 3MM™ paper system (N ¼21) Routine system
Range RCP
Median RCP
Average RCP
7 STD
ITLC-SG/MEK ITLC-SG/saline Aggregate RCP
94.5–98.8 98.3–99.8 92.9–98.4
97.8 99.2 97.0
97.3 99.2 96.5
1.26 0.44 1.57
3MM™ paper system
Range RCP
Median RCP
Average RCP
7 STD
3MM/acetone:dichloromethane 3MM/saline Aggregate RCP
96.0–99.8 97.4–99.9 93.7–97.8
98.0 98.9 96.9
98.0 98.8 96.8
0.75 0.73 0.97
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tetrahydrofuran solvents required, and an important safety concern that these solvents might be harmful to operators. In a paired t-test comparison of the two sets of aggregate RCP values, there wasn’t a statistically significant difference (p ¼0.549). Interestingly, when corresponding values of the separate strips for impurity detection of free pertechnetate and reduced hydrolyzed technetium are compared, statistical significance is seen (p¼ 0.038 and p¼ 0.019, respectively, for the saline and organic solvent systems). However, the two component systems seem to counterinfluence the aggregate RCP. Routine strips developed in 0.9% saline gave a somewhat higher average value of 99.2% 70.43, to the Whatman 3MM™ paper strips’ 98.8% 70.70. On the other hand, routine strips developed in organic solvent gave somewhat lower average values of 97.3% 71.26 compared to Whatman 3MM™ paper strips’ 98.0% 70.75. Therefore, it appears that although the aggregate average RCP values for 99mTc-tetrofosmin in the two systems are near-identical (96.5 and 96.8%), the Whatman 3MM™ slightly but consistently underweights the impurity reduced hydrolyzed technetium and slightly but consistently overweights the free pertechnetate impurity. An argument can be made that these values give slightly different impression of the dosimetry and biological distribution of the product when injected in a patient, but this concerns are not significant and these observations do not have an overage impact on quality of the preparation or its clinical use.
alternative. Also, for 99mTc-sestamibi a small but consistent undervaluing of the RCP was obtained with the Tec-Control™ strips when compared to the paired ITLC-SG results. This difference appears to be consistent with that obtained by a similar t-testing of the sample results presented by Zimmer, 2004. Formal manufacturer procedures included in many package inserts specify ITLC-SG or other systems for the determination of radiochemical purity, This study validates Tec-Control™ and Whatman 3MM™ paper strip systems as alternatives to the established ITLC-SG quality control systems. It is important that institutions satisfy themselves by method validation or other evaluation processes that alternative procedures achieve similar or clearly defined results, and in this case the results supported a transitioning primarily to the Tec-Control™ system, supplemented with the Whatman 3MM™ system specifically for QC of 99mTctetrofosmin.
Acknowledgements Staff at the Radiopharmacy, Health Sciences Centre for administrative and technical assistance. References
4. Conclusion When silica gel-impregnated glass fiber sheets (ITLC-SG, Pall Life Sciences, MI) were withdrawn from the market, there was strong motivation to identify, evaluate and adopt if possible alternative QC procedures. Miniaturized Tec-Control™ chromatography strips and Whatman 3MM™ paper strips were investigated to obtain indications on their accuracy, reliability and reproducibility. The results obtained were compared to those obtained using routine ITLC-SG quality control. The examination of the pre-cut Tec-Control™ strips can be considered to have been successful for all products with the exception of 99mTc-tetrofosmin. The Whatman 3MM™ paper strips also proved to be successful. Both sets of media were not only accurate and reliable, but they were much faster and easier to use than the routine strips. They show largely similar and comparable chromatographic (Rf) profiles and radiochemical purity values. The results obtained for Tec-Control™ strips support other reported results (Ivanov, 2010; Ivanov et al., 2010; Monteiro et al., 2009; Zimmer, 2004), and support the determination that the strips are good substitutes for ITLC-SG. However, though the Tec-Control™ and Whatman 3MM™ paper strips are both good substitutes, Tec-Control™ strips take only a fraction of the time to develop compared to the Whatman 3MM system and are therefore a better (more convenient) product. The respective total development times for each strip were Whatman 3MM™ (7–9 min), TecControl™ ( 1 min) and ITLC-SG (10–15 min). Also, because the Tec-Control™ strips were unsuccessful in the QC of 99mTctetrofosmin, this study proposes that Whatman 3MM™ paper strips are the preferred alternative to the routine strips for this preparation. A majority of the products were tested with sufficient replicates that it was possible to perform paired t-tests of the comparative samples. In the case of 99mTc-sestamibi, the solvent system never was able to migrate to the top of the Whatman 3MM™ paper strips and this system does not appear to be a viable
Amin, K.C., Sagar, P., Doke, A., Saha, G.B., 2011. An alternative method for determining the radiochemical purity of 99mTc-tetrofosmin. J. Nucl. Med. Technol. 39 (1), 51–54. Barnes, P., Jenkins, M., Baldock, C., Lutkin, J.E., Burwod, R.J., Jeyakumar, T., 1996. Comparison of the TEC-Control (TM) test kit and the ‘Chen method’ for determination of radiochemical purity (RCP) for MAG3. Nucl. Med. Commun. 17 (4), 267. Billinghurst, M.W., Jette, D., 1979. Colloidal particle-size determination by gel filtration. J. Nucl. Med. 20, 133–137. Billinghurst, M.W., Eckert, K., Mang’era, K, 2004. Alternative quality control for technetium-99m IDA complexes. Appl. Radiat. Isot. 61 (6), 1151–1155. Eggert, L.A., Dick, M.D., Mahoney, D.W., Olson, J.P., Werner, G.L., Hung, J.C., 2010. A rapid radiochemical purity testing method for 99mTc-tetrofosmin. J. Nucl. Med. Technol. 38, 81–84. Hammes, R., Joas, L.A., Kirschling, T.E., Ledford, J.R., Knox, T.L., Nybo, M.R., Sterzinger, J.J., 2004. A better method of quality control for 99mTctetrofosmin. J. Nucl. Med. Technol. 32 (2), 72–78. Hung, J.C., Wilson, M.E., Brown, M.L., Gibbons, R.J., 1991. Rapid preparation and quality control method for technetium-99m-2-methoxy isobutyl isonitrile (technetium-99m-sestamibi). J. Nucl. Med. 32, 2162–2168. Ivanov, Z., Guilfoyle, R., Bokor, I., 2010. Alternatives to instant thin layer chromatography-silica gel (ITLC-SG) for use in the radiochemical purity measurement of reconstituted ‘Cold Kit’ {99m}Tc-radiopharmaceuticalsAust. N. Z. Nucl. Med. 41 (1), 6–8. Ivanov, Z., 2010. Results of the Quality Assurance Testing Program for Radiopharmaceuticals 2009. Australian Radiation Protection and Nuclear Safety Agency Technical Report, 153 Millar, A.M., O’Brien, L.M., Beattie, L.A., Craig, F., 2010. An evaluation of paper chromatography for measuring the levels of radiochemical impurities in 99mTc medronate injection. J. Label. Compd. Radiopharm. 53, 11–14. Monteiro, E.G., de Almeida, É.V., Ramos, M.P.S., Alves, E.V., Benedetti, S., Mengatti, J., Fukumori, N.T.O., Matsuda, M.M.N., 2009. Comparison of chromatography systems for radiochemical purity determination of lyophilized reagents labeled with technetium-99m. In: International Nuclear Atlantic Conference—INAC. Rio de Janeiro, RJ, Brazil, September 27–October 2, 2009. 〈http://www.ipen.br/ biblioteca/2009/inac/15119.pdf〉, accessed May 8, 2013. Ponto, J.A., 2011. Evaluation of alternative methods for radiochemical purity testing of indium-111 capromab pendetide. J. Am. Pharm. Assoc. (2003) 51 (3), 359–362. Saha, GB., 2010. Fundamentals of Nuclear Pharmacy, sixth ed. Springer, New York Zimmer, A.M., 2004. Miniaturized Chromatography Procedures for Radiopharmaceuticals: 2004 update. Biodex Corp Manual, 2004 Zlata, I., Robert, G., Ilonka, B., 2010. Alternatives to instant thin layer chromatography-silica gel (ITLC-SG) for use in the radiochemical purity measurement of reconstituted ‘cold kit’ 99mTc-radiopharmaceuticals. Aust. N. Z. Nucl. Med. 41 (1), 6–8.
Contents lists available at ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Evaluation of alternative rapid thin layer chromatography systems for quality control of technetium-99m radiopharmaceuticals Kennedy Mang0 era a,b,c,n, Derek Wong a,c, David Douglas b, Kellie Franz a, Taddese Biru a a
Radiopharmacy, Health Sciences Centre, GC219-820 Sherbrook Street, Winnipeg, MB, Canada R3A1R9, Radiology, Faculty of Medicine, University of Manitoba, GA216-820 Sherbrook Street, Winnipeg, MB, Canada R3T 2N2 c Faculty of Pharmacy, University of Manitoba, 750 McDermot Avenue, Winnipeg, MB, Canada R3E 0T5 b
H I G H L I G H T S
Whatman 3MM™ paper and Tec-Control™ systems validated as ITLC-SG alternatives. Full solvent migration in Tec-Control™ was not reproduced for 99mTc-tetrofosmin. Systems are comparable to ITLC-SG, Tec-Control™ is more conservative for 99mTc-sestamibi. Tec-Control™ ( 1 min) and Whatman 3MM™ (7–9 min) are faster than ITLC-SG (10–15 min). Overall, Tec-Control™ is preferred but Whatman 3MM™ recommended for 99mTc-tetrofosmin.
art ic l e i nf o
a b s t r a c t
Article history: Received 16 September 2012 Received in revised form 1 October 2013 Accepted 16 December 2013 Available online 8 January 2014
Whatman 3MM™ and Tec-Control™ systems were evaluated as ITLC-SG alternatives for 99mTcradiopharmaceuticals. They compare well in accuracy and reproducibility, and are faster and more convenient than ITLC-SG. Tec-Control™ radiochemical purity values for 99mTc-sestamibi were more conservative than ITLC-SG. Full solvent migration was not reproduced for 99mTc-tetrofosmin in TecControl™, and for this Whatman 3MM™ is preferred. Developing times were 10–15 min, 7–9 min and 1 min for ITLC-SG, Whatman 3MM™ and Tec-Control™, respectively. Overall, Tec-Control™ strips are preferred due to speed and ease of use. & 2013 Elsevier Ltd. All rights reserved.
Keywords: Quality control ITLC-SG Whatman 3MM™ Tec-Control™ Technetium Radiopharmaceuticals
1. Introduction Silica gel-impregnated instant thin layer chromatography (ITLC-SG) strips have been in widespread use in the quality control of radiopharmaceuticals for over three decades (Saha, 2010). The major advantages of a validated ITLC analytical process have been the speed of the test and its relative simplicity and inexpense. The primary vendor of ITLC-SG strips (Pall Life Sciences, Ann Arbor, MI) discontinued supply in 2008, with severe implications for everyday radiopharmacy practice, eased later by availability of the product from a different vendor (Agilent, product SGI0001). This discontinuation followed that of polysilicic acid-impregnated ITLC n Corresponding author at: Radiopharmacy, Health Sciences Centre, GC219-820 Sherbrook Street, Winnipeg, MB, Canada R3A1R9. Tel.: þ 1 204 7873540; fax: þ1 204 787 1313. E-mail address: [email protected] (K. Mang0 era).
0969-8043/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.apradiso.2013.12.016
strips in 2004/5, which were widely used for the quality control of 99mTc-dimercaptosuccinic acid and other 99mTc-iminodiacetic acid (IDA) complexes. Subsequently, an alternative solid extraction procedure for the 99mTc-mebrofenin was developed and validated, applicable to other 99mTc-IDAs (Billinghurst et al., 2004). A number of groups have also reported on alternative quality control systems to replace ITLC-SG systems, although most of the literature is applicable to single radiopharmaceutical products (Amin et al., 2011; Eggert et al., 2010; Ponto, 2011; Zlata et al., 2010). The central radiopharmacy at Winnipeg Health Sciences Centre supplies and provides services to five hospitals affiliated to the Winnipeg Regional Health Authority and prepares a variety of radiopharmaceuticals. QC operations would therefore have been severely impacted by the discontinuation of the commercial ITLCSG strips and there was strong motivation to identify and validate QC tests alternative to the routine ITLC-SG systems (Table 1),
K. Mang0 era et al. / Applied Radiation and Isotopes 86 (2014) 57–62
58
Table 1 Routine chromatography systems used at the radiopharmacy. Product
Media
Manufacturer and model
Solvent
99mTc-DTPA
ITLC-SGa,b
Pall Corp. 61886
99mTc-MDP
ITLC-SGa,b
Pall Corp. 61886
Methylethyl ketone 0.9% Saline Methylethyl ketone 0.9% Saline 0.9% Saline Ethanol Methylethyl ketone Methylethyl ketone Ethyl Acetate
a
99mTc-SbS colloid 99mTc-Sestamibi 99mTc-DMSA 99mTc-Sulfur colloid 99mTc-Tetrofosmin a b c
ITLC-SG Aluminum Oxide IB-Fc ITLC-SGa ITLC-SGa ITLC-SGa
Pall Corp. 61886 J.T. Baker 4467-02 Pall Corp. 61886 Pall Corp. 61886 Pall Corp. 61886
Developed in a 22 6 cm glass cylindrical tank. Developed in a 30 30 cm rectangular chamber. Developed in a 18 12 cm rectangular chamber.
Table 2 ITLC chromatography systems based on Tec-Control™ miniaturized strips. Product
Media
Strips color-codes
Solvent
Developing vial (mL)
99mTc-DTPA
Whatman 31 ET ITLC-SG
Red
Acetone
10
Black
Distilled water Acetone
99mTc-MDP
Whatman 31ET ITLC-SG
99mTc-SbS colloid 99mTc-Sestamibi
ITLC-SG Whatman 31ET 99mTc-DMSA Whatman 31ET 99mTc-Sulfur colloid Whatman 31ET 99mTc-Tetrofosmin Whatman 1
Red Black
Product
Solvent
99mTc-MDP
Methylethyl ketone 0.9% Saline Methylethyl ketone 0.9% Saline Ethyl Acetate Acetone: Dichloromethane
99mTc-SbS colloid 99mTc-Sulfur colloid 99mTc-Tetrofosmin 10
10 5
Yellow
Distilled water 0.9% Saline Ethyl acetate Acetone
Red
Acetone
10
Teal
Ethyl acetate
5
Dark green Pink
Table 3 Chromatography systems based on Whatman 3MM™ strips.
10
technetium radiolabelled complex after subtraction of tested radiochemical impurities (free pertechnetate, reduced hydrolyzed technetium). Preparation and evaluation of the effect of vial size and shape on RCP following the Tec-Control procedures was determined using two vial sizes, 5 mL (Biodex, Shirley, NY) and 10 mL (Greer Laboratories, Lenoir, NC). 2.2. Quality control procedures
including the commercial options Tec-Control™ chromatography (Biodex, Shirley, NY; Table 2) (Barnes et al., 1996; Zimmer, 2004) and Whatman 3MM™ paper system (Table 3). Tec-Control systems are available in color-coded strips for specific radiopharmaceuticals. These systems were compared with the standard ITLC-SG procedures routinely applied at our radiopharmacy.
2. Materials and methods 99mTc-sodium pertechnetate was eluted from Tecknekow™ generators (Mallinckrodt, St. Louis, MO). 2.1. Preparation of 99mTc-radiopharmaceuticals Commercial kits of diethylene triaminopentaacetic acid (DTPA, DraxImage), methylene phosphonate (MDP, DraxImage), sestamibi (Covidien and Cardiolite, Lantheus Medical), dimercaptosuccinic acid (DMSA, GE Healthcare) and sulfur colloid (GE Healthcare) were reconstituted according to manufacturer product inserts to make 99mTc-radiopharmaceuticals. Procedures for preparation of 99mTc-tetrofosmin (Myoview, GE Healthcare) were adopted from the manufacturer0 s product insert, but higher activities (up to 25 GBq 99mTc-pertechnetate) were used in reconstitution. 99mTcantimony sulphide (99mTc-SbS) colloid was prepared using inhouse kit formulations and following established procedures (Billinghurst and Jette, 1979). For all preparations, the pass limits for radiochemical purity is 90%, calculated as the net percentage
All spotting of product samples onto the strips were performed using 28 G, 1 cm3 insulin syringes (VanishpointTM, Retractable Inc., Little Elm, Texas). Various operators were involved in conducting the QC tests, but in all cases the same person performed each comparative test set. For detection of radiochemical species, the chromatography strips were run on an in-house TLC scanner equipped with a 5 cm NaI(Tl) detector and connected to an Eagle Plus MCA system and a Genie-200 Basic software (Canberra, Concord, Ontario). The comparative time delay to full development of the spotted strips by the relevant solvent in the chamber was determined for the two alternative strip systems and compared to the development times of the routine ITLC-SG systems. (a) Routine ITLC-SG and thin layer chromatography (TLC) Table 1 summarizes the routine ITLC-SG chromatography systems for each product. The strips (1.5 20 cm) were cut from 20 20 cm impregnated glass fiber sheets (Pall Life Sciences, Ann Arbor, MI). Product was spotted 2.5 cm from the bottom and developed in a closed 22 6 cm glass cylindrical tank or a closed 30 30 cm developing chamber, which contained the appropriate developing solvent. When solvent migrated to a mark 2.5 cm from the top, the strips were immediately removed, left to dry (except where solvent was saline), and then wrapped in plastic film and analyzed on the TLC counter. 99mTc-sestamibi analysis requires use of aluminum oxide IB-F chromatography strips (J.T. Baker, Phillipsburg, NJ, USA). The pre-cut 2.5 7.5 cm strips were solvated with a drop of
K. Mang0 era et al. / Applied Radiation and Isotopes 86 (2014) 57–62
ethanol, spotted with the product and air-dried before developing in ethanol in an enclosed 18 12 cm rectangular developing chamber. The strips were removed when the solvent front was 1 cm from the top, wrapped in plastic film and analyzed on the TLC counter. (b) Tec-Control™ chromatography The Tec-Control™ chromatography systems evaluated for the various radiopharmaceuticals are summarized in Table 2. The procedures using Tec-Control™ chromatography strips were adopted from literature supplied by the vendor (Zimmer, 2004). The color-coded miniaturized strips are supplied pre-cut as 6 0.7 cm strips (7 0.7 cm for 99mTc-sestamibi). A small drop of product was spotted on the marked origin line (1 cm from the bottom) and the strips developed in their appropriate solvents in either “off-the-shelf” 10 mL vials (Greer Laboratories, Lenoir, NC), or the suggested 10 mL and 5 mL vials (Biodex, Shirley, NY). Once the strips were developed to the marked solvent front line (1 cm from the top), they were immediately removed, wrapped in plastic film and analyzed on the TLC counter. (c) Whatman 3MM™ paper Table 3 summarizes the chromatography systems based on Whatman 3MM™ paper that were evaluated for various radiopharmaceuticals. Whatman 3MM™ paper (Whatman Inc., Clifton, NJ) was obtained as 20 20 cm sheets, and cut into 1.5 7.5 cm strips. Evaluating these strips involved spotting the product 1 cm from the bottom and allowing them to develop to a mark 1 cm from the top. The strips were developed in an enclosed 18 12 cm rectangular chamber, filled with the appropriate solvent. The strips were immediately removed when solvent front was reached, wrapped in plastic film and analyzed on the TLC counter. Chromatography strips developed in acetone:dichloromethane were air-dried before being wrapped in plastic film and analyzed on the TLC counter.
59
Table 4 Radiochemical purity values (%) obtained for 99mTc-DTPA preparations analyzed using routine ITLC-SG and miniaturized Tec-Control™ systems. 99mTc-DTPA—Routine system versus miniaturized system (N ¼7) Routine system
Range RCP
Median RCP
Average RCP
7 STD
ITLC-SG/MEK ITLC-SG/saline Aggregate RCP
99.6–99.9 99.3–99.9 98.9–99.8
99.9 99.8 99.7
99.8 99.7 99.5
0.12 0.25 0.37
Miniaturized system
Range RCP
Median RCP
Average RCP
7 STD
Tec-Control™/acetone Tec-Control™/water Aggregate RCP
98.4–99.9 98.4–99.7 96.5–99.6
99.3 99.3 98.6
99.3 99.0 98.3
0.51 0.75 1.06
Table 5 Radiochemical purity values (%) obtained for 99mTc-MDP preparations analyzed using routine ITLC-SG and miniaturized Tec-Control™ systems (A), and radiochemical purity values (%) obtained for 99mTc-MDP preparations analyzed using routine ITLC-SG and Whatman 3MM™ paper strips (B). (A) 99mTc-MDP—Routine system versus miniaturized system (N ¼ 17) Routine system
Range RCP
Median RCP
Average RCP
7STD
ITLC-SG/MEK ITLC-SG/saline Aggregate RCP
95.6–99.7 95.8–99.6 91.4–98.9
98.8 99.2 98.2
98.4 98.4 96.8
1.35 1.35 2.42
Miniaturized system
Range RCP
Median RCP
Average RCP
7STD
Tec-Control™/acetone Tec-Control™/water Aggregate RCP
88.2–99.9 96.5–99.8 87.9–99.7
99.7 99.7 99.4
98.8 99.5 98.3
2.79 0.83 2.82
(B) 99mTc-MDP—Routine system versus 3MM™ paper system (N ¼19) Routine system
Range RCP
Median RCP
Average RCP
7STD
ITLC-SG/MEK ITLC-SG/saline Aggregate RCP
96.2–99.8 85.2–99.5 83.9–98.4
98.6 98.7 96.9
98.3 97.8 96.1
0.90 3.20 3.17
3. Results and discussion
3MM™ paper system
Range RCP
Median RCP
Average RCP
7STD
The effectiveness of the miniaturized Tec-Control and the Whatman 3MM chromatography paper strips was studied against routine ITLC-SG QC chromatography methods and using routine 99mTc-kit preparations at the central radiopharmacy. Paired t-test calculations in runs with adequate number of samples are all twotailed. Developing times of each set of strips were monitored. Generally, the routine ITLC-SG strips took the longest to develop at approximately 10 min to 15 min. The Whatman 3MM™ paper strips took 7 min to 9 min to develop, whereas the miniaturized Tec-Control™ strips developed in about a minute. The proposed systems also show largely similar and comparable chromatographic (Rf) profiles to the corresponding ITLC-SG methods.
Tec-Control™/acetone Tec-Control™/water Aggregate RCP
95.7–99.9 91.8–99.1 90.8–98.1
98.8 97.9 95.3
98.4 97.2 95.6
0.95 1.97 2.04
3.1. 99mTc-DTPA Assessing % RCP of 99mTc-DTPA requires a two-strip system to demarcate impurity levels of free pertechnetate and reduced hydrolyzed technetium. The results summarized in Table 4 compare corresponding ITLC-SG and Tec-Control™ single strip procedures and compare the aggregate RCP of 99mTc-DTPA after accounting for both impurities. The routine ITLC-SG strips developed in MEK (demarcating reduced hydrolyzed technetium levels) gave an average value of 99.8%7 0.12 while the corresponding miniaturized strips developed in acetone gave an average value of 99.3% 70.51. Routine
strips developed in 0.9% saline (demarcating free pertechnetate levels) gave average value of 99.7% 70.25 while the corresponding miniaturized strips developed in distilled water gave average value of 99.0% 70.25. The miniaturized strips gave an average RCP for 99mTc-DTPA of 98.3% 7 1.06, a value that is comparable to the aggregate RCP of 99.5% 70.37 determined using routine strips. 3.2. 99mTc-MDP As for 99mTc-DTPA, two-strip systems were used to determine separately the levels of the two expected impurities in 99mTcMDP and an aggregate RCP was calculated for the preparation. Results are presented in Table 5. Routine ITLC-SG strips developed in MEK to demarcate colloid impurity measured a purity average of 98.4% 7 1.35 while the corresponding miniaturized strips developed in acetone measured 98.8% 72.79. For free pertechnetate impurity, routine strips developed in 0.9% saline measured an average RCP of 98.4% 71.35 (note: coincidentally, values are identical to MEK above but data set is different) and the miniaturized strips developed in distilled water measured 99.5% 70.83. Routine strips determined
60
K. Mang0 era et al. / Applied Radiation and Isotopes 86 (2014) 57–62
an aggregate RCP of 96.8% 7 2.42 while the miniaturized strips gave an average aggregate RCP of 98.3% 72.82. Paired two-tailed t-test results showed that the overall RCP values for 99mTc-MDP preparations are not statistically different for the miniaturized strips compared to the routine ITLC-SG strips used in daily QC procedures (p¼ 0.588). Paradoxically, although the miniaturized system gave a higher average RCP value, for the one sample where there was a quality significance, this system gave an overall RCP of 87.9% (product fails QC), compared with 92.5% (product passes QC) obtained for the ITLC-SG system. The one result appears to be an anomaly. Whatman 3MM™ paper strips were also compared to the routine strips (Table 5B). The routine strips developed in organic solvent detected a RCP of 98.3 70.90%, whereas Whatman 3MM™ paper strips had an average % RCP of 98.4% 70.93. The results of both sets of strips correlated well with each other. The routine strips developed in saline detected a RCP of 97.8% 73.20, whereas the Whatman 3MM™ paper strips gave 97.2% 71.97. Again, the % RCP results correlated well with each other. Millar et al. (2010) evaluated five Whatman papers (1CHR, 31ET, 3MM, 4CHR and 54SFC) using methyl ethyl ketone for detecting 99mTc-pertechnetate and using sodium acetate solution (136 g/L) for detecting reduced hydrolysed technetium impurities. They found the highest resolutions with 1CHR, 4CHR and 54SFC and erroneously high levels of colloidal and 99mTc-pertechnetate when using 1CHR and 4CHR. They proposed 54SFC paper as a suitable alternative to ITLC-SG for measuring radiochemical impurities in 99mTc-MDP preparations. Paired two-tailed t-test for our results showed that the overall RCP values for 99mTc-MDP preparations are statistically significantly lower for the Whatman 3MM™ strips compared to the routine ITLC-SG strips used in daily QC procedures (p¼ 0.025). The significance of results of paired t-test values for the individual systems delineating the impurities free pertechnetate (saline is solvent) and RHT colloid (organic solvent) were mixed, with values of p ¼0.014 and p ¼0.708, respectively. The somewhat generally lower overall RCP values obtained for the miniaturized system had a practical quality significance in one sample where an overall RCP of 87.9% was obtained, compared with 92.5% obtained for the ITLCSG system. For this sample, the ‘fail0 and ‘pass’ conclusions for quality are therefore conflicting, and the miniaturized system appears to provide more conservative results for quality when compared to ITLC-SG analysis.
3.3. 99mTc-Antimony sulphide (99mTc-SbS) colloid Comparative quality control for 99mTc-antimony sulphide (99mTc-SbS) colloid was performed using all three systems, the routine ITLC-SG, the miniaturized Tec-Control™, and the Whatman 3MM™ paper strips. All three systems are a one-strip procedure that distinguishes 99mTc-SbS from free pertechnetate (which migrates to the solvent front). Results comparing routine ITLC-SG and miniaturized Tec-Control™ systems are presented in Table 6A. The routine strips detected an average RCP of 98.8% 70.62 while the miniaturized strips detected a RCP of 98.7% 7 1.60. These values are nearidentical. Results comparing routine ITLC-SG and Whatman 3MM™ paper strips are presented in Table 6B. The routine strips show an average % RCP of 97.6 71.21 Whatman 3MM™ strips give a RCP of 98.0% 7 2.47. Overall, paired t-test analysis shows that the results obtained for the routine and miniaturized strips are similar, with no significant statistical difference (p ¼ 0.872). A similar similarity without statistical significance is seen in the pairing of the routine and Whatman 3MM™ strips samples (p ¼0.611).
Table 6 Radiochemical purity values (%) obtained for 99mTc-SbS Colloid preparations analyzed using routine ITLC-SG and miniaturized Tec-Control™ systems (A), and radiochemical purity values (%) obtained for 99mTc-SbS Colloid preparations analyzed using routine ITLC-SG and Whatman 3MM™ paper strips (B). (A) 99mTc-SbS colloid—Routine system versus miniaturized system (N ¼11) Routine system
Range RCP
Median RCP
ITLC-SG/saline
97.5–99.5
98.6
Miniaturized system
Range RCP
Median RCP
Tec-Control™/saline
95.0–99.9
98.8
Average RCP 7 STD 98.8
0.62
Average RCP 7 STD 98.7
1.60
(B) 99mTc-SbS colloid—Routine system versus 3MM™ paper system N ¼ 13 Routine system
Range RCP
Median RCP
ITLC-SG/saline
95.9–99.5
97.3
3MM™ paper system
Range RCP
Median RCP
3MM/saline
91.0–99.9
98.5
Average RCP 7 STD 97.6
1.21
Average RCP 7 STD 98.0
2.47
Table 7 Radiochemical purity values (%) obtained for 99mTc-Sestamibi preparations analyzed using routine TLC and miniaturized Tec-Control™ systems. 99mTc-Sestamibi—Routine system versus miniaturized system (N ¼ 21) Routine system
Range RCP
Median RCP
Average RCP
7 STD
TLC/ethanol
90.9–98.5
97.9
97.5
1.59
Miniaturized system
Range RCP
Median RCP
Average RCP
7 STD
Tec-Control™/saline
88.1–98.9
97.2
96.7
2.20
3.4. 99mTc-Sestamibi When comparing the routine plates and miniaturized strips for quality control of 99mTc-sestamibi (Table 7, N ¼21), the mean and median RCP obtained using the miniaturized strips (96.7 and 97.2%) are very similar to those obtained by the routine TLC/ ethanol plates (97.5 and 97.9%, respectively). These results obtained from the routine and miniaturized strips were therefore consistent with one another. However, a notable observation for 99mTc-sestamibi results is that for one sample the product passed the limits for quality when measured by the routine procedure (RCP¼90.9%) but failed the limits when determined using the miniaturized strips (RCP¼88.1%). Additionally, paired two-tailed t-test results showed that although the individual paired RCP values were consistently close, they were also consistently highly statistically different (p¼ 0.0005). The RCP values were consistently lower for the miniaturized strips compared to the routine TLC/ethanol system. Although the difference between the two systems is small, the results straddle either side of the pass margin of 90% and this result has clear clinical significance. Overall, the miniaturized system provided more conservative results for quality for 99mTc-sestamibi analysis. In studying a variety of 99mTc-radiopharmaceuticals and comparing ITLC-SG with two alternative procedures (Whatman 3MM™ and Whatman 1MM™) with 99mTc-sestamibi being the common one with our study, Monteiro et al. (2009) obtained near-identical RCP values between the ITLC-SG and the Whatman systems for 99mTc-sestamibi (N¼4, two replicates of two preparations). They monitored the preparations at 30 min, 60 min and 240 min.
K. Mang0 era et al. / Applied Radiation and Isotopes 86 (2014) 57–62
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compare very well to the routine strips, with values of 99.5% 70.38 and 98.6% 71.17, respectively. Results comparing the routine ITLC-SG and Whatman 3MM strips for % RCP are presented in Table 9B. The routine strips detected a RCP of 96.7% 7 0.63, whereas the Whatman 3MM™ paper strips detected a RCP of 97.4% 70.81. Overall, the % RCP values obtained from the Whatman 3MM™ paper strips are consistent with those of the routine ITLC-SG strips.
Paired t-test evaluation of RCP values reported in a similar comparative study by Zimmer, (2004) also show consistently slightly lower values for the miniaturized system compared to the routine TLC system (N ¼13); and that this difference is statistically significant (p¼ 0.033). Their results have mean RCP values for the routine and the miniaturized systems of 97.4% 70.84 and 96.9 71.46, respectively. 3.5. 99mTc-DMSA
3.7. 99mTc-Tetrofosmin
A comparison of corresponding ITLC-SG and Tec-Control™ results obtained for QC of 99mTc-DMSA are summarized in Table 8. Values obtained for RCP are high and virtually identical for the two systems, with average RCP values of 99.5% 70.65 and 99.3% 70.54, respectively.
Our evaluation of 99mTc-Tetrofosmin using the miniaturized chromatography method was unsuccessful as the solvent-phase would only develop two-thirds up the strip (up to the vial’s lip), and would remain there indefinitely. The ethyl acetate eventually evaporated from the vial after approximately fifteen minutes of developing time. Evaluation of Whatman 3MM™ on preparations of 99mTcTetrofosmin was as a two-strip system, and the chromatography strips were developed in both acetone:dichloromethane (DCM) and 0.9% saline in order to separately quantify the impurity levels of free pertechnetate and reduced hydrolyzed technetium, respectively. Results are presented in Table 10. Routine ITLC-SG strips developed in acetone:DCM gave an average aggregate RCP of 96.5% 71.57 whereas Whatman 3MM™ paper strips showed a very similar average aggregate RCP of 96.8% 70.95. Other alternative procedures have been suggested for quality control of 99mTc-tetrofosmin (Hammes et al., 2004). Eggert et al. (2010) evaluated a method adopted from the QC of 99mTc-sestamibi (Hung et al., 1991) that uses solvent saturation pads (Krackeler Scientific, Inc.) as stationary phase and a 1:1 chloroform:tetrahydrofuran mixture as mobile phase. Although they found this alternative method more accurate, they cited a burdensome preparation and refrigeration process for the combination of chloroform and
3.6. 99mTc-Sulfur colloid Results comparing routine ITLC-SG and miniaturized TecControl strips for 99mTc-Sulfur Colloid preparations are presented in Table 9A. The % RCP values given by the miniaturized strips Table 8 Radiochemical purity values (%) obtained for 99mTc-DMSA preparations analyzed using routine TLC and miniaturized Tec-Control™ systems. 99mTc-DMSA—Routine system versus miniaturized system (N ¼ 5) Routine system
Range RCP
Median RCP
ITLC-SG/MEK
98.4–99.9
99.9
Miniaturized system
Range RCP
Median RCP
Tec-Control™/acetone
98.4–99.8
99.5
Average RCP
7STD
99.5
0.65
Average RCP
7STD
99.3
0.54
Table 9 Radiochemical purity values (%) obtained for 99mTc-Sulphur Colloid preparations analyzed using routine TLC and miniaturized Tec-Control™ systems (A), and radiochemical purity values (%) obtained for 99mTc-Sulphur Colloid preparations analyzed using routine ITLC-SG and Whatman 3MM™ Paper System (B). (A) 99mTc-Sulphur colloid—Routine system versus miniaturized system (N ¼ 7) Routine system
Range RCP
Median RCP
Average RCP
7 STD
ITLC-SG/MEK Miniaturized system Tec-Control™/saline
96.7–99.5 Range RCP 96.5–99.6
98.2 Median RCP 99.0
98.6 Average RCP 99.5
1.17 7 STD 0.38
(B) 99mTc-Sulphur colloid—Routine system versus 3MM™ paper system (N¼ 12) Routine system
Range RCP
Median RCP
Average RCP
7 STD
ITLC-SG/MEK 3MM system 3MM/saline
95.9–98.1 Range RCP 96.1–99.3
96.8 Median RCP 97.4/97.5
96.7 Average RCP 97.4
0.63 7 STD 0.81
Table 10 Radiochemical purity values (%) obtained for 99mTc-tetrofosmin preparations analyzed using routine ITLC-SG and Whatman 3MM™ paper strips. 99mTc-Tetrofosmin—Routine system versus 3MM™ paper system (N ¼21) Routine system
Range RCP
Median RCP
Average RCP
7 STD
ITLC-SG/MEK ITLC-SG/saline Aggregate RCP
94.5–98.8 98.3–99.8 92.9–98.4
97.8 99.2 97.0
97.3 99.2 96.5
1.26 0.44 1.57
3MM™ paper system
Range RCP
Median RCP
Average RCP
7 STD
3MM/acetone:dichloromethane 3MM/saline Aggregate RCP
96.0–99.8 97.4–99.9 93.7–97.8
98.0 98.9 96.9
98.0 98.8 96.8
0.75 0.73 0.97
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tetrahydrofuran solvents required, and an important safety concern that these solvents might be harmful to operators. In a paired t-test comparison of the two sets of aggregate RCP values, there wasn’t a statistically significant difference (p ¼0.549). Interestingly, when corresponding values of the separate strips for impurity detection of free pertechnetate and reduced hydrolyzed technetium are compared, statistical significance is seen (p¼ 0.038 and p¼ 0.019, respectively, for the saline and organic solvent systems). However, the two component systems seem to counterinfluence the aggregate RCP. Routine strips developed in 0.9% saline gave a somewhat higher average value of 99.2% 70.43, to the Whatman 3MM™ paper strips’ 98.8% 70.70. On the other hand, routine strips developed in organic solvent gave somewhat lower average values of 97.3% 71.26 compared to Whatman 3MM™ paper strips’ 98.0% 70.75. Therefore, it appears that although the aggregate average RCP values for 99mTc-tetrofosmin in the two systems are near-identical (96.5 and 96.8%), the Whatman 3MM™ slightly but consistently underweights the impurity reduced hydrolyzed technetium and slightly but consistently overweights the free pertechnetate impurity. An argument can be made that these values give slightly different impression of the dosimetry and biological distribution of the product when injected in a patient, but this concerns are not significant and these observations do not have an overage impact on quality of the preparation or its clinical use.
alternative. Also, for 99mTc-sestamibi a small but consistent undervaluing of the RCP was obtained with the Tec-Control™ strips when compared to the paired ITLC-SG results. This difference appears to be consistent with that obtained by a similar t-testing of the sample results presented by Zimmer, 2004. Formal manufacturer procedures included in many package inserts specify ITLC-SG or other systems for the determination of radiochemical purity, This study validates Tec-Control™ and Whatman 3MM™ paper strip systems as alternatives to the established ITLC-SG quality control systems. It is important that institutions satisfy themselves by method validation or other evaluation processes that alternative procedures achieve similar or clearly defined results, and in this case the results supported a transitioning primarily to the Tec-Control™ system, supplemented with the Whatman 3MM™ system specifically for QC of 99mTctetrofosmin.
Acknowledgements Staff at the Radiopharmacy, Health Sciences Centre for administrative and technical assistance. References
4. Conclusion When silica gel-impregnated glass fiber sheets (ITLC-SG, Pall Life Sciences, MI) were withdrawn from the market, there was strong motivation to identify, evaluate and adopt if possible alternative QC procedures. Miniaturized Tec-Control™ chromatography strips and Whatman 3MM™ paper strips were investigated to obtain indications on their accuracy, reliability and reproducibility. The results obtained were compared to those obtained using routine ITLC-SG quality control. The examination of the pre-cut Tec-Control™ strips can be considered to have been successful for all products with the exception of 99mTc-tetrofosmin. The Whatman 3MM™ paper strips also proved to be successful. Both sets of media were not only accurate and reliable, but they were much faster and easier to use than the routine strips. They show largely similar and comparable chromatographic (Rf) profiles and radiochemical purity values. The results obtained for Tec-Control™ strips support other reported results (Ivanov, 2010; Ivanov et al., 2010; Monteiro et al., 2009; Zimmer, 2004), and support the determination that the strips are good substitutes for ITLC-SG. However, though the Tec-Control™ and Whatman 3MM™ paper strips are both good substitutes, Tec-Control™ strips take only a fraction of the time to develop compared to the Whatman 3MM system and are therefore a better (more convenient) product. The respective total development times for each strip were Whatman 3MM™ (7–9 min), TecControl™ ( 1 min) and ITLC-SG (10–15 min). Also, because the Tec-Control™ strips were unsuccessful in the QC of 99mTctetrofosmin, this study proposes that Whatman 3MM™ paper strips are the preferred alternative to the routine strips for this preparation. A majority of the products were tested with sufficient replicates that it was possible to perform paired t-tests of the comparative samples. In the case of 99mTc-sestamibi, the solvent system never was able to migrate to the top of the Whatman 3MM™ paper strips and this system does not appear to be a viable
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