A system for high performance liquid chromatography of 99mTc-compounds with on-line radiometric detection and data processing

hr. f. Appl. Rariiar. /sol. Primed in Great Britain

Vol. 36,

No. 5. pp. 349-355.

1985

0020-708X:85 53.00 + 0.00 Pergamon Press Lrd

A System for High Performance Liquid Chromatography of gg”Tc-Compounds with On-line Radiometric Detection and Data Processing G. J. DE GROOT’, H. A. DAS and C. L. DE LIGNY2 ‘Netherlands Energy Research Foundation (ECN), P.O. Box 1, 1755 ZG Petten, The Netherlands and ‘Laboratory for Analytical Chemistry, State University, Utrecht Croesestraat 77a, 3522 AD Utrecht, The Netherlands (Received 20 November 1984)

A system for HPLC with on-line 7 activity (two channels) and u.v.-detection is described, including software for the on-line data processing with a microcomputer. It was developed for the analysis of WmTc-diphosphonates. Examples of some of its features are gjven using the separation of ““Tc and “‘Sn labeled Tc(Sn)EHDP complexes with ion-pair-chromatography.

1. Introduction

99mTc has received growing attention recently.“) Originally the predominant separation technique was gel permeation chromatography (GPC), which was also applied in our earlier investigationso-‘) The application of high performance liquid chromatography (HPLC) to 99mTc-diphosphonate preparations was first reported in 19g0.(4)This paper and further publications(‘.s) emphasized the inherent advantages of speed and specificity in product control. To realize these potential advantages it is desirable to have on-line radiometric and optical detection as well as on-line data processing facilities available. The present text describes the system for HPLC with on-line y-activity and u.v.-detection and the accompanying on-line data processing, that we developed for the separation and identification of WmTc-diphosphonates.

High-frequency filtering of the data, to reduce the influence of the noise, is optional. Filtering has three advantages: data can be processed by a relatively simple peak detection program, minor peaks can be detected and samples with a low level of radioactivity can be analysed. Read-out is possible as a graphical display and as a list of peak-positions, widths, net areas and their standard errors. Computation is automatic or interactive, to ensure control by the experimenter. The peak-areas are measured for windows around the main peaks of 99mTc(= 141 keV) and of a second isotope, if necessary. These areas are transformed into fractional amounts by using the total content of the radiogram for the radionuclide involved. The total content of the on-line radiogram is occasionally checked by off-line counting of an identical aliquot as used in the HPLC system. It can easily be derived that the ratio (total on-line)/(total off-line) for the same volume of sample is given by:

2. Principle

r = (E,,/E,R)*(V~~V)*~/T,R

A promising HPLC-mode for the separation of radiopharmacia is reversed phase ion-pair chromatography. In the present system this method is used. In addition, the system has the following features. The system records on-line the U.V. signal and the 7 activity of 99mTc and, if necessary, of a second radionuclide (e.g. as a label for the reducing agent, “3Sn-“3mIn for Sn(II), 48V for V(II1) and “Fe for Fe(II)].

where E,, and E,, are the counting efficiencies for on- and off-line measurements, Vr the volume of the on-line detection cell (mL), v the flow-rate in the on-line detection mode (mL/min), To, the off-line counting time (min). Once the E,,,lE,,ratio has been measured for the radionuclides involved at a constant window setting of the detector, one can use the off-line mode for checks on the total recovery.

The separation and identification of the compounds which are formed in labeling diphosphonates with

349

(1)

G. J. DE GROCT et al.

350

A special case is offered by the label ‘13Sn (t, 1= ll9d)- ‘13”In (t, 2 = 100 min). In the off-line mode the daughter-product is measured, whereas for on-line detection one has to rely on the parent. Due to the large difference in the abundances of the relevant y-lines, off-line detection is by far superior here. 3. Equipment and Chemicals The system is presented in Fig. 1. It consists of: -Solvent delivery module, (Beckman, model 112). -Injector, (Beckman, model 2 10). -HPLC analytical column (4 x 220 mm), (Brownlee (OD-244)) reversed phase C-18, 5~ spheri. -Variable wavelength detector, (Beckman, mode1 165). -3 x 3 in. well-type NaI(Tl) detector, well-size: 16 mm 4 x 52 mm with a 35 p L gamma-detection cell (coiled Teflon tube). Spectroscopy amplifier, (Canberra, model 1417). Two single channel analyzers, (Ortec, model 406A). -Fraction collector (LKB, mode1 221 I). -Chart recorder, (Linear, mode1 595). -Microcomputer (Apple IIe). Additional hardware: Two disk-drivers, (Siemens). Data acquisition/control card, modified to accept two additional digital signals in two timechannels, (Adalab, Interactive Microware Inc.). 128 K ram expansion card, (Vergecourt Ltd, Ramex 128). Clock card (Magister Software), also used as the timer for the data acquisition/control card. Extended 80-column card for Apple He. -Matrix

The chemicals used are of p.a. quality; EHDP is synthesised according to Ref. (6). The 99mTcis obtained from a WMo/99”T~ generator of 100-200 mCi, (Stercow 99m-DRN 4332, Byk-Mallinckrodt CIL B.V.). 4. Data Processing Software was made for processing of the three data lines: two from activity counting and one from the u.v.-absorption measurement. It is written mainly in Basic. The program is interactive and in a menu-like form (the basic structure of the menu was derived from the Applesoft Tutorial (ASL2003)). All routines are accessible from the menu; they are stored in the 128 K ram card. Since the memory of the Apple can contain only one routine at a time, the routines are transferred to the memory sequentially. To speed up the procedure and for transferring the most important variables from one routine to another, they are compiled with TASC (Microsoft Inc.) using the “Commonblock” option. The software contains the following routines: -Start -Menu -Preparation conditions of sample -Data collection: (1). run conditions (2). execution -Data filter (FFT-filter) -Run conditions report and plot of data -Peak detection/background-correction and report of the results Description

printer, (Epson RX-80).

of the most important

routines:

Eluent a

r-4

Injector

I

HPLC

1

1 collector

Fig.

1.

Layout of the system for on-line radio-HPLC.

1

An HPLC system for *“Tc compounds

351

Start

Peak-detection

The Start routine transfers the programmes from disk to the 128 K ram card and initialises the “commonblocks”.

After adequate filtering a simple peak-detection routine is in most cases sufficient. The routine used determines Start, Top and End of peaks by detecting relative increments and decrements in the value of successive data points. The threshold for Start and End is variable. A reference peak-width was determined empirically for the separation system used. The expected peakwidth for a certain position in the radiogram was calculated by using the proportional relationship between retention time and peak-width. If a further check is performed by calculating the net contents of the peak found and comparing it with its standard deviation according to:

iMenu The Menu routine gives access to all routines mentioned except ‘Start”. It has the following options: Main menu:

(1)data acquisition (submenu( 1)) (2)data processing (submenu(2))

Submenu ( 1):

(1)enter preparation conditions of sample (2)enter experimental conditions and start data acquisition

Submenu (2):

(1)display data from memory (2)display data from disk (3)load data from disk (4)Iilter data (5)report data (sample preparation and run conditions + plot) (6)perform peak detection and report results (7)perform automatic processing of data in memory

Data acquisition

Two digital (radioactivity) and one analog (u.v.) signal are collected by a modified interface card. The preset sampling frequency is timed by the clockcard. The sampling is performed by a separate machinelanguage routine which is loaded and activated by the data acquisition program. The preset sampling frequency is determined by setting the duration of the run (in minutes). The program chooses the minimal sampling time (in 0.5 s time intervals) for which the amount of data points does not exceed 1024 (max. data points/run). The run can be broken off at any time without losing the data already collected. During the run the display can be switched between a plot and a numerical display. FFT jilter

The principle of the FFT-filter is to calculate a frequency spectrum of the data and to eliminate the higher frequencies. The frequencies of the noise in the radioactivity measurements are generally higher than those of the peaks. They are eliminated by a combined hamming/rectangular-filter and after inverse transformation, a filtered radiogram is obtained. The FFT-filter program allows the choice of either automatic determination of the filter-parameters or manual processing with the aid of a plot of the frequency-spectrum. The FFT-subroutine used in our FFT-filter program is a modified version of a routine from Schutte.“)

PI - BI > 3*[(std. PI)’ + (std. BI)‘]“’ PI BI std. PI std. BI

= = = =

(2)

gross peak-content background under peak standard deviation PI standard deviation BI

The routine is designed to detect a maximum of 20 peaks in a radiogram. If necessary, the program automatically increases the threshold until the number of peaks found does not exceed 20. It supplies retention times, peak-widths, heights and peak contents. Reasonably adequate peak contents of slightly overlapping peaks can be obtained by choosing the option for overlap correction. This is based on measured heights and retention times of the individual peaks and on the peak content of the group of overlapping peaks: PC(J) = FH(J)*TP(J)/TP(

I)]*TPC/TPH

TPH = sum [(PH(J)*TP(J)/TP( I)] PC(J) PH(J) TP(J) TP(I)

(3) (4)

= = = =

corrected peak-content net-peak-height retention time of peak retention of time of first peak of group of overlapping peaks TPC = total net-content of group of overlapping peaks TPH = sum of heights of overlapping peaks corrected for differences in retention times.

Radiograms with severely overlapping peaks fall outside the present scope, as they require processing by a curve-fitting program. Another optional feature is correction of the baseline. The baseline can be constructed with at most ten straight lines. Every slope and height within the frame can be interactively chosen with the aid of a plot of the data. Entering off-line data

A separate program is used for entering off-line data from the Apple keyboard and storing them on

352

G. J. DE GROOT et al.

disk in a data file than can be processed by the system.

(e) Before injection, TBA and EtAc were added to the sample in the same concentration as in the eluent to avoid artefacts. 23 PL was injected.

5. Results The procedure and its limitations are demonstrated for the case of the separation of Tc(Sn)EHDP complexes, labeled with 99mT~and “3Sn-‘i~“In. The 99”Tc(Sn)EHDP was prepared by stannous sulfate reduction Of WmT~O; in the presence Of diphosphonatel” as follows:

.4 print-out of the experimental conditions. Plots of the radiograms and u.v.-signal. Print-outs of the results of the peak detection of the radioactivity data,

(a) Stannous sulfate (1 .Omg) and 2.0 mg of sodium acetate was dissolved in 15 mL 0.005 M EHDP deaerated aqueous solution, leading to 0.0003 M SnSO, and 0.001 M NaAc. (b) A spike of “‘Sn(i-0.1 PCi) in 6N HCl was added and the pH was lowered to 6.0. After an equilibration time of 10 min, a spike of 53 mCi 99mTc0; in 0.45% NaCl was added. (c) After a reaction time of 1.5min the pH was raised to 7.2-7.3; the preparation was filtered through a 0.5 p Millipore filter and stored under nitrogen. (d) The eluent [a modification of the eluent used in Ref. (l)] consisted of: 0.005 IM EHDP, 0.003 M tetra butyl ammonium bromide (TBA), 0.0003 M SnSO,, 0.001 M NaAc, 1.0% ethyl acetate (EtAc) and pH = 7.2-7.3.

A part of the obtained plots of the radiogram is shown in Figs 2-5. Figures 2a and b show the results of processing (including filtering) of on-line and off-line measurements, respectively, on 99mT~. Five major complexes are found. In general the resolution of peaks of on-line detection is improved by a factor 1.2-1.5 in comparison with off-line detection and moreover the results are obtained in a much shorter time. Figure 3 shows the results of processing of off-line data on “‘“In (The daughter of “‘Sn). Five major Sn-EHDP complexes are found. From the sharpness and the symmetry of the peaks it can be concluded that no appreciable exchange occurs between the radioactive Sn-EHDP complexes and the Sn(II) in the eluent, in the short separation time (30-45 min).

(a)

Data are presented in three parts:

g9mTC

16

15

(b)

25

30

g9m Tc

0

I

5

10

15 Time

Fig. 2. Radiogram

20

( min 1

Time

of Tc(Sn)EHDP

20

25

30

( min 1

complexes. (a) Results of on-line off-line counting of WmTc.

counting

of *“‘Tc. (b) Results

of

353

An HPLC system for W”Tc compounds ,I,”

,”

5

0

10

15 Time

20

The effect of the data-filtering is shown in Fig. 4. The signal to noise ratio improvement by using a FFT-filter depends mainly on the number of data points sampled per peak. In the example given, with approximately 60 data points sampled per peak, the S/N ratio is increased by a factor 4.5. Even without a large S/N ratio improvement (with a 10~ sampling rate), filtering is useful: the only noise that remains has a width (frequency) similar to that of the data peaks. which enables data processing with a relatively simple peak detection program. Figure 5 illustrates the option of baseline correction.

30

25

( mtn1

An example of a print-out of the results of the peak detection routine is shown in the appendix.

Fig. 3. Radiogram of Sn-EHDP (and Tc(Sn)EHDP) complexes. Results of off-line counting of “‘51-+““In.

FFT 0

2

4

6

6

Time

10

12

14

Cmm1

(b)

Id)

99m Tc IN”

0

2

4

6

6

Time

1min

10

12

FFT

14

I

Fig. 4. The different steps of a FFT-filtering procedure. (a) Raw data. (b) frequency-spectrum. (c) A filtered spectrum: hamming from 20th harm. Rectangular from 41st harm. (d) Filtered data.

354

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J.

DE

GROOT et al.

‘4 typical time-budget HPLC procedure is:

(a)

for the present

Event

per data

file:

Start of the system Separation Filtering data Reporting/plotting of data

Baseline correction Peak detection

on-line

Duration

(min)

1 45 2-5

l-2 1-2 2-3 +55

Total (b)

6. Conclusions The described system is suitable for fast processing of on-line ;: activity data with the aid of a microcomputer. To the authors’ knowledge there is no commercial software available that performs this task.

i__

-

ji___&

Fig. 5. An example of interactive baseline correction. (a) Uncorrected radiogram with interactively chosen baseline. (b) Corrected radiogram.

Acknowledgemenu-The authors wish to thank R. Moor for writing the machine-language subroutine for the data acquision and Drs H. van Nieuwkerk for the fruitful discussions about computer programming.

References The present HPLC procedure enables separations to be done within 45 min. This constitutes a considerable time-gain compared to normal GPCseparations where 10-12 h are needed.“’ This gives the opportunity to study changes in the composition of the reaction mixture with time. If these occur, the results of HPLC analysis are more representative for the composition of the reaction mixture at the time where it is used for scanning, than the results of GPC are. If data have to be obtained by off-line counting, this will take several hours of counting time and the additional time needed for transformation into suitable format for further processing with a computer.

1. Srivastava S. C., Meinken G. E., Richards L. A., Ford L. A and Benson W. R. Proc. 3rd World Congress of Nucl. Med. and Biol., Paris, Vol II, p. 1631 (Pergamon

Press, Oxford, 1982). 2. Van den Brand J. A. G. M., Das H. A., Dekker B. G. and de Li_my C. L. ht. J. Appl. Radiat. Isor. 32, 637 (1981).

3. Van den Brand J. A. G. M., Dan H. A., Dekker B. G. and de Li_my C. L. ht. J. Appl. Radial. Isot. 33, 917

(1982). 4. Pinkerton T. C., Heineman W. R. and Deutsch E. Anal. Gem. 52, 1106 (1980). 5. Pinkerton T. C., Ferguson D. L., Deutsch E., Heineman W. R. and Libson K. Inr. J. Appl. Radiar. hot. 33,907 (1982).

6. Castronovo F. P. J. Nucl. Med. 15, 127 (1974). 7. Schutte H. H. Kluwer Sofiware-reeks (1983), Deventer (The Netherlands).

355

An HPLC system for *“Tc compounds

APPENDIX Print-out of the results of the peak detection routine of the data shown in Fig. Za Report date: 05-NOV-84 Data file: COM3 * 200684/37- 122 Peak

Start (mitt)

End (min)

1)

5.07- 5.87

2)

5.87- 7.73 7.73- 8.30 8.30 8.63 8.63- 9.43 9.43-l 0.07 10.07-I 1.10 ll.lO-II.53 11.53-14.03 14.03-16.50 16.90-17.90 17.90-19.47 22.23-25.37 25.70-26.57 27.00-28.87 28.87-29.90

3) 4) 5) 6) :; 9) 10) If> 12) 13 14 15) 16)

(99”Tc)

Number of data: 900

Top (min)

Content k SD (C/2 s)

Height counts

Width (min)

Plate NR

5.52 6.77 7.92 8.54 8.95 9.71 10.94 11.35 13.13 14.56 17.63 18.16 24.10 26.35 27.23 29.23

5544 * 88 36132 + 219 3525+71 1587 5 48 4266 + 78 3324 1: 69 5445 i: 89 2451 k 60 71573 _S308 33504 & 214 2336 k 62 3297 & 75 22229 k 179 1918 k 58 3193 + 77 1581 k 55

444 1864 253 167 187 185 186 201 2147 1080 84 85 505 81 84 58

0.31 0.29 0.26 0.18 0.35 0.33 0.55 0.20 0.88 0.41 0.51 0.95 1.02 0.23 0.26 0.57

1238 2207 3712 9198 2661 3368 1599 12487 894 5065 4733 1473 7136 -__ 50570 44133 10690

Background = 15.7 (SD = 2.39/N = 152) NET-Tot. number of counts = 209032 (counts are corrected for decay since preparation time!) Peak :; 3) 4) 5) 6) ;; 9) IO) If> 12) 13 14 15) 16)

Tr (min)

aa

% Total

Type

5.52 6.77 7.92 8.54 8.95 9.71 10.94 II.35 13.13 14.56 17.63 18.16 24.10 26.35 27.23 29.23

*o. *0.21 15 r0.26 0.29 *0.30 *0.34 80.40 *0.42 80.50 *0.56 80.70 *0.73 RI.00 1.10 *I.14 1.24

17.29 2.65

vs’ B- Vf sv V-V V-V V-V V-V V- Vf V-V VS- sv+ V-V V-V V-V V- sv V-V V-V V-B

1.69 0.76 2.04 1.59 2.60 1.17 34.24 16.03 1.12 1.58 10.63 0.92 1.53 0.76

Threshold peak detection+peak-begin/peak-end: 0.8 x SD of background. ‘Standard base-width Ref-peak (R): 3.86 min (MB = 0.16). Void vol. = 2.3 mL. Peaks marked with “)” or “)” have overlap with others. Peak-contents are not corrected for overlap.

(Type-code) (B: (V: baseline) valley) (’ : top) (S: shoulder) (T: tailing) (+ : broadened)