Thin-layer chromatography of technetium radiopharmaceuticals

Stationary phases:

ITLC-SG Instant thin-layer chromatography, silica gel, Pal Gelman Laboratory

ITLC-SA Instant thin-layer chromatography, silicic acid, Gelman (No longer available)

3MM Whatman 3MM chromatography paper

No 1 Whatman No 1 chromatography paper

silica gel Silica gel 60, e.g. Merck

alumina aluminium oxide, Bakerflex

cellulose cellulose, e.g. Merck

Mobile phases:

butanone = 2-butanone = methyl ethyl ketone = MEK

1 M sodium acetate = 82 mg/mL anhydrous sodium acetate

or 136 mg/mL sodium acetate trihydrate

0.1 M citrate = 21 mg/mL monosodium citrate dihydrate

1 M ammonium acetate = 77 mg/mL ammonium acetate

mixtures of volatile solvents should be made freshly each day

Radiopharma-ceutical	Stationary phase	Mobile phase	Rf RH-Tc	Rf TcO_₄	Rf Tc-bound
^99mTc-pertechnetate	ITLC-SG	acetone or saline	0.0	1.0	-
^99mTc-MDP	ITLC-SG or 3MM	acetone	0.0	1.0	0.0
^99mTc-MDP	ITLC-SG	1 M sodium acetate or saline	0.0	1.0	1.0
^99mTc-DTPA	ITLC-SG or 3MM	Acetone	0.0	1.0	0.0
^99mTc-DTPA	ITLC-SG or 3MM	saline	0.0	1.0	1.0
^99mTc-colloid	ITLC-SG or 3MM	acetone or saline	0.0	1.0	0.0
^99mTc-DMSA	3MM	acetone	0.0	1.0	0.0
^99mTc-DMSA	ITLC-SA	butanol acidified with 0.3 M HCl	0.0	0.9	0.5
^99mTc-MAA	ITLC-SG or 3MM	acetone or saline	0.0	1.0	0.0
^99mTc-pyrophosphate	ITLC-SG or 3MM	acetone	0.0	1.0	0.0
^99mTc-pyrophosphate	ITLC-SG	water	0.0	1.0	1.0
^99mTc-HSA	ITLC-SG or 3MM	acetone	0.0	1.0	0.0
^99mTc-HSA	ITLC-SG strip should be pre-saturated with human serum albumin and dried	ethanol-ammonia-water (2:1:5)	0.0	1.0	1.0
^99mTc-HIG	ITLC-SG or 3MM	acetone, saline, or 0.1 M citrate	0.0	1.0	0.0
^99mTc(V)-DMSA	ITLC-SG	butanone	0.0	1.0	0.0
^99mTc(V)-DMSA	ITLC-SG	saline	0.0	1.0	1.0
^99mTc(V)-DMSA	silica gel	butanol-acetic acid- water (3:2:3)	0.0	0.8	0.5
^99mTc-IDAs	ITLC-SA	20% sodium chloride	0.0	1.0	0.0
^99mTc-IDAs	3MM spot must be dry	butanone	0.0	0.9	0.0
^99mTc-IDAs	ITLC-SG	water or 50% acetonitrile	0.0	1.0	1.0
^99mTc-sestamibi	Alumina Pre-spot with ethanol; do not allow spot to dry	ethanol	0.0	0.0	1.0
^99mTc-tetrofosmin	ITLC-SG spot must be dry	acetone-dichloromethane (35:65)	0.0	1.0	0.5
^99mTc-MAG3	ITLC-SG	ethyl acetate-butanone (3:2)	0.0	1.0	0.0
^99mTc-MAG3	ITLC-SG	50% acetonitrile	0.0	1.0	1.0
^99mTc-exametazime	ITLC-SG	butanone	0.0	1.0	1.0
^99mTc-exametazime	ITLC-SG	saline	0.0	1.0	0.0
^99mTc-exametazime	No 1	50% acetonitrile	0.0	1.0	1.0
^99mTc-sulesmurab (Leukoscan)	ITLC-SG or 3MM	acetone, saline, or 0.1 M citrate	0.0	1.0	0.0
^99mTc-depreotide (Neospect)	ITLC-SG	saturated solution of sodium chloride	0.0	1.0	0.0
^99mTc-depreotide (Neospect)	ITLC-SG	1 M ammonium acetate-methanol (1:1)	0.0	1.0	1.0

Substitutions:

in most cases, 2-butanone (methyl ethyl ketone, MEK) can be substituted for acetone

in most cases, water can be substituted for saline

in most cases, Whatman No 1 can be substituted for Whatman 3MM paper

ACD can be substituted for 0.1 M citrate

Thin-layer chromatography of other radiopharmaceuticals

Radiopharma-ceutical	Stationary phase	Mobile phase	Rf free	Rf bound
¹⁴C-urea	cellulose	butanol-water-acetic acid (12:5:3)	?	0.6
^123/131I-hippuran	silica gel	chloroform-acetic acid (9:1)	0.0	0.2-0.3
^123/131I-MIBG	silica gel	ethyl acetate-ethanol (1:1)	0.6	0.0
¹¹¹In-DTPA	ITLC-SG	10% ammonium acetate- methanol (1:1)	0.1	1.0
¹¹¹In-octreotide	ITLC-SG	0.1 M citrate buffer pH 5	1.0	0.0
¹⁸F-FDG	silica gel	acetonitrile-water (95:5)	0.0	0.6
¹²³I- ioflupane	ITLC-SG spot must be dry	chloroform-methanol (9:1)	0.0	1.0
¹²³I-iomazenil	silica gel	ethyl acetate-ammonium hydroxide (200:1)	0.0	0.7
¹²³I-iomazenil	silica gel	chloroform-acetic acid- water (65:35:5)	0.0	0.3
¹³¹I-iodocholesterol	silica gel	chloroform-ethanol (1:1)	0.0	0.66

Solid-phase extraction cartridge methods (e.g. Sep-Pak)

General procedure:

1. Pre-wet (“activate”) cartridge with 2-5 mL ethanol or methanol.

2. Prepare cartridge with 2-10 mL of preparation solvent.

3. Place a drop of the radiopharmaceutical in the inlet of the cartridge.

4. Elute sequentially with 2-10 mL quantities of eluates A, B, C and collect each in a separate tube; after the last eluate, force air through the cartridge to dry it.

5. Place the cartridge in another tube for measurement of residual activity.

6. Measure the activity in each tube in an ionisation chamber.

7. Calculate radiochemical purity as per table.

Radiopharma-ceutical	Type of cartridge	Prepar-ation solvent	A	B	C	D	Purity
^99mTc-sestamibi	alumina N	0.5 mL ethanol	10 mL ethanol	cartridge residue			A/total
^99mTc- sestamibi	C18	2 mL saline	2 mL saline	5 mL ethanol	cartridge residue		B/total
^99mTc- tetrofosmin	C18	2 mL saline	2 mL saline	5 mL ethanol	cartridge residue		B/total
^99mTc- tetrofosmin	silica	5 mL saline then 1 mL air	10 mL methanol-water (70:30) over 2 minutes	cartridge residue			B/total
^99mTc- tetrofosmin	silica	5 mL saline then 1 mL air	10 mL methanol-water (70:30) over 2 minutes	10 mL methanol-saline (80:20)	cartridge residue		B/total
^99mTc-MAG3	C18	10 mL 1 mM HCl	10 mL 1 mM HCl	10 mL 50% ethanol	cartridge residue		B/total
^99mTc-MAG3	C18	10 mL 1 mM HCl	5 mL 1 mM HCl	5 mL 0.5% ethanol in PB	10 mL 7% ethanol in PB	cartridge residue	C/total
^99mTc-exametazime	C18	5 mL saline	5 mL saline	cartridge residue			B/total
^99mTc-exametazime	C18	5 mL saline	5 mL saline	5 mL ethanol	cartridge residue		B/total
¹¹¹In- octreotide	C18	10 mL water	5 mL water	5 mL methanol	cartridge residue		B/total
¹²³I-ioflupane	C18	5 mL water	5 mL water	5 mL ethanol	cartridge residue		B/total
^123/131I-MIBG	C18	5 mL water	5 mL water	10 mL PB-THF (3:1)	cartridge residue		B/total
^123/131I-MIBG	C18	5 mL water	5 mL 10 mM NaOH	cartridge residue			B/total

Preparation of reagents:

1 mM HCl (0.001 M HCl) = 1 mL conc HCl per litre of distilled water.

PB for MAG3 = 0.01 M (10 mM) sodium phosphate buffer pH 6

Prepare 100 mL 0.01 M monosodium phosphate solution (NaH₂PO₄). Prepare 20 mL 0.01 M disodium phosphate solution (Na₂HPO₄). Add 10 mL disodium phosephate solution to 100 mL monosodium phosphate solution. pH should still be below 6. Add disodium phosphate solution dropwise until pH of 6 is obtained.

PB for MIBG = 0.1 M (100 mM) monosodium phosphate (NaH₂PO₄)

THF = tetrahydrofuran

10 mM NaOH (0.01 M NaOH) = 0.4 g dissolved in 1 litre of distilled water

or dilute 1 mL 1 M NaOH with 99 mL distilled water

Cartridges can be re-used after decay of radioactivity

^99mTc-exametazime Extraction Method

1. Prepare a 10 ml test tube containing 3 ml of ethyl acetate and 3 ml of 0.9% w/v sodium chloride solution.
2 Add several drops of ^99mTc-exametazime (immediately after reconstitution).
3 Cap the tube and mix on a vortex mixer for 1 min.
4 Allow the tube to stand for 1 min to allow the two phases to separate.
5 Remove the top layer using a pipette into another test tube.
6 Measure the activities in each layer.
7 Calculate the % lipophilic complex as follows:-
% Primary complex = Activity in the ethyl acetate layer x 100 (should be greater than 80 %)
Total activity in both layers