CC BY-NC-ND
0NUKLEONIKA 2016;61(1):69-74 doi: 10.1515/nuka-2016-0015
ORIGINAL PAPER - REGULAR PAPER
S DE GRUYTER
New amino bisphosphonate compound Thaer Assaad
for skeletal imaging: Comparison study with methylenediphosphonic acid (MDP) and (1-hydroxyethane-1,1-diyl) diphosphonic acid (HEDP)
Abstract. A novel bisphosphonate derivative (1-aminoethane-1,1-diyl)diphosphonic acid (AEDP) has been prepared and successfully labeled with 99mTc at high labeling yields. The in vivo biodistribution of 99mTc-AEDP has been investigated and compared with two reference compounds Tc-99m methylene diphosphonate (99mTc-MDP) and Tc-99m (1-hydroxyethylidene) diphosphonate (99mTc-HEDP). The biodistribution studies have demonstrated a high uptake of the radiotracer 99mTc-AEDP in the bone and a rapid clearance from the blood (such as the two technetium-labeled bone imaging agents in current use: 99mTc-MDP and 99mTc-HEDP). Additionally, the scintigraphic images of 99mTc-AEDP in normal rats have revealed high selective skeletal uptake.
Key words: aminoethylidenediphosphonic acid • biodistribution • bisphosphonates • bone imaging agent • radiolabeling
T. Assaad
Radioisotopes Division,
Radioisotopes Department,
Atomic Energy Commission of Syria (AECS),
P. O. Box 6091, Damascus, Syrian Arab Republic,
Tel.: +963(11) 213 2580, Fax: +963(11) 611 2289,
E-mail: cscientific@aec.org.sy
Received: 10 August 2015 Accepted: 18 January 2016
Introduction
Technetium-99m radiopharmaceuticals play an important role in wide range of applications in nuclear medicine. Bone imaging agents are among the first developed 99mTc-radiopharmaceuticals and the most widely used radiopharmaceuticals in diagnostic nuclear medicine [1]. Bisphosphonates (BPs) are synthetic organic compounds characterized by a P-C-P backbone structure. They are chemically stable analogues of the endogenous metabolites, inorganic pyrophosphates. The biological effects of BPs on calcium metabolism were originally ascribed to their physicochemical effects to impede the dissolution of hydroxyapatite crystals [2-4].
Because of their inhibitory effect on the bone resorption, various types of BPs are used in bone scanning, and provide an effective way of diagnosis of primary bone cancer, metastatic bone disease, Paget's disease, osteoporosis, bone trauma, etc. Several 99mTc-labeled BPs have been synthesized, such as methylenediphosphonic acid (MDP) [2], (1-hydroxyethane-1,1-diyl)diphosphonic acid (HEDP) and hydroxyl methylene diphosphonate (HMDP), (Fig. 1), and used in nuclear medicine for both diagnosis and treatment purposes [5-14].
To continue the previous efforts to find better bone-imaging agents, we report in this manuscript the synthesis of a new bisphosphonate compound, AEDP, and investigate its radiolabeling abilities with 99mTc. Subsequently, we have performed preliminary in vivo studies in rats and compared the results with those obtained using two reference
oh oh oh oh o=p-oh o=p_oh o=p—oh o=p_oh
h_c_h ho_c_h h3c—c—oh h3c—c—nh2
o=p—oh o=p_oh o=p-oh o=p_oh
oh mdp
oh hmdp
oh hedp
oh aedp
o nh2o
Oh Ch30h
Fig. 1. Bisphosphonate derivatives. MDP: methylenedi-phosphonic acid; HMDP: hydroxyl methylene diphospho-nate; HEDP: (1-hydroxyethane-1,1-diyl)diphosphonic acid; AEDP: (1-aminoethane-1,1-diyl)diphosphonic acid.
compounds, 99mTc-MDP and 99mTc-HEDP in order to determine whether 99mTc-AEDP is convenient as a bone-seeking agent.
Materials and methods
All chemical reagents and solvents were of commercial quality and used as received. NMR spectra were acquired using a Bruker Bio spin 400 spectrometer (400 MHz for *H, 100 MHz for 13C). Chemical shifts (8) were expressed in ppm relative to tetramethyl silane (TMS) as an internal standard. Melting point (MP) determination was performed using a digital melting point instrument from Stuart model SMP3. Infrared spectra were recorded as KBr pellets in the range 4000-400 cm-1 using an FTIR-JASCO 300E. ITLC (instant thin-layer chromatography) measurement was carried out using Whatman No. 3 strips (Sigma Chemical Company, USA) and radioactivity counted in a gamma scanner (Raytest mini GITA, Model BGO-V-detector) equipped with NaI(Tl) detector and single channel analyzer. MDP and HEDP kits were provided as commercial available kits produced by Atomic Energy Commission of Syria. Bone scan was performed with a gamma camera (Siemens Signature, Duel head, Damascus, Syria). Microanalysis was performed using a EURO EA analyzer. X-ray powder diffraction (XRD) patterns were obtained using a Stoe Stadi-P diffractometer with monochromatic CuKa1 radiation (X = 1.5406 A) selected using an incident-beam curved-crystal germanium Ge(111) monochromator, using the Stoe transmission geometry (horizontal set-up) with a linear position-sensitive detector (PSD).
Experimental
Synthesis of (1-aminoethane-1,1-diyl)diphosphonic acid (AEDP)
To a mixture of acetonitrile (150 ml), phosphorous acid (16.8 g, 0.2 mol) in glacial acetic acid (10 g, 0.167 mol) was added dropwise phosphoryltrichlo-ride (51.7 g, 0.334 mol) at 55-65°C. The resulting mixture was stirred for 24 h at 70-75°C. The reaction mixture was cooled down to 60-65°C, and then water (150 ml) was added slowly. The reaction temperature was then increased to 90-100°C and
Scheme 1. Synthesis of (1-aminoethane-1,1-diyl)diphos-phonic acid (AEDP).
maintained for the next 4-6 h. The reaction mixture was cooled down to 0-5°C and stirred for 3 h. The solid product was separated by filtration, washed with water, and finally, with methanol to produce the corresponding product, in 77% yield. Appearance: white powder (Scheme 1), MP = 279.8-276.6°C.
Spectroscopic data of AEDP: 'H-NMR (D2O): 81.46-1.53 (t, 3H, CH3). 13C-NMR (D2O): 817.5 (1C, CH3), 52.6-55.1 (1C, C-CH3). 31P-NMR (D2O): 813.39 (2P, P-OH). IR (KBr, v cm-1): 3448-3236 (OH, NH2), 2338 (P-H), 1602 (O = P-O-H), 1142 (P = O). Analytical data for (AEDP): Found: C, 11.30; H, 4.41; N, 6.48; Calculated C, 11.72; H, 4.42; N, 6.83.
Synthesis of (1-hydroxyethane-1,1-diyl)diphosphonic acid (HEDP)
A mixture of (5.13 g, 62.56 mmol) phosphorous acid and (7.74 g, 75.07 mmol) dried acetic anhydride was refluxed for 4 h at 105°C (Scheme 2). At the same temperature, water vapor was bubbled through the mixture until the distillate became almost free of acid. The reaction mixture was concentrated in a rotary evaporator to yield crystals of HEDP monohydrate. The solid product was separated by filtration and washed with acetone to produce the corresponding product, in 90% yield, MP = 106.3-106.6°C.
Spectroscopic data of HEDP: 1H-NMR (D2O): 51.63 (t, 3H, CH3), 54.88 (s, H2O). 13C-NMR (D2O): 5 19.16 (1C, CH3), 68.83-71.75 (1C, C-CH3). 31P-NMR (D2O): 519.84 (2P; P-OH). IR (KBr, v cm-1): 3444-3551 (OH), 2394 (P-H), 1602.6 (O = P-O-H), 1142.4 (P = O).
Powder X-ray diffraction analysis of AEDP
The compound crystallizes as a fine white powder, therefore, a laboratory powder X-ray diffraction data was used for phase identification. The powder X-ray diffraction data were collected at room temperature with a STOE transmission STADI-P diffractometer using CuKa1 radiation (X = 1.54060 Â) selected with an incident-beam curved-crystal Ge(111) monochromator with a linear PSD. The pattern was scanned over the angular range 5.0-95.0° (28). For phase identification, a Crystallography Open
H3PO3 + (CH3CO)2O
O CH3O
ho—LLLoh .
0h 0h 0h
Scheme 2. Synthesis of (1-hydroxyethane-1,1-diyl)diphos-phonic acid (HEDP). MDP: methylenediphosphonic acid.
Fig. 2. The final Rietveld plot for (1-aminoethane-1,1-diyl)diphosphonic acid (AEDP).
Database (COD) interfaced by the program QualX
[15] was used. This program reports that the crystal structure of this compound was published by Tsaryk et al. [16] as a second triclinic polymorph of (1-ammonio-1-phosphonoethyl)phosphonate. Moreover, Rietveld analysis of the powder pattern for this compound (Fig. 2) indicates that the asymmetric unit of this compound, C2H9NO6P2, contains one molecule as a zwitterion. The N atom of the amino group is protonated and one of the phosphonic acid groups is deprotonated. Bond lengths and angles are similar to those obtained from single crystal data
[16]. H atoms involved in these hydrogen bonds are located at inversion centers. These bonds and additional O-H...O and N-H...O hydrogen bonds interlink the molecules, giving a three-dimensional supramolecular structure (Fig. 3).
Preparation of kits
375 mg of AEDP or HEDP was dissolved in 15 ml double distilled water in a vial. 312 |l of freshly prepared aqueous solution of SnCl2-2H2O in nitrogen purged (400 mg SnCl2-2H2O/0.5 ml concentrated HCl/5 ml H2O) was added into the vial. The pH was adjusted to 7.5 with sodium hydroxide solution. The final solution was adjusted with water to 25 ml and the resulting solution was sterilized by filtration through a cellulose ester filter (0.22 |im). Aliquots of
1 ml were transferred to glass vials and lyophilized for 24 h. The lyophilized vials were sealed under vacuum and stored in a refrigerator at 4°C.
Radiolabeling of AEDP, HEDP, and MDP with 99mTc
Radiolabeling of the kit involves initial warming up of the vial to room temperature followed by the addition of 370-740 MBq (10-28 mCi) of freshly eluted 99mTcO4- in 2 ml of normal saline and finally, incubation of the vial for 15 min at room temperature. Radiochemical purity was determined by ITLC as follows: 5 |l of the prepared compound was spotted on 10 cm Whatman No. 3 strips (Sigma Chemical Company, USA). The strips were then run with acetone and saline solution as mobile phases. After developing, the radioactivity was counted by a gamma scanner (Raytest mini GITA, Model: BGO-V-detector) equipped with NaI(Tl) detector and a single channel analyzer. By using acetone as the mobile phase, reduced 99mTc and 99mTc-complexes remained near the point of spotting with Rf values <0.2, while free 99mTcO4- moved towards the solvent front. By using saline solution as another mobile phase, 99mTc-complexes and 99mTcO4-moved towards the solvent front with Rf values >0.9, whereas reduced 99mTc remained at the point of spotting (Figs. 4 and 5).
Fig. 3. The molecular structure of (l-aminoethane-1,1--diyl)diphosphonic acid (AEDP) obtained by Rietveld refinement.
Fig. 4. TLC pattern of 99mTc-AEDP complex in acetone.
rmijimiinijiHii 200 300
r i rt p I r 1"| 11U | r 1 n | r r r» J ri r 11 n T»1 n 1 n 1 r 11 r rn rn 1 »0 « 0 70.0 80.0 90.0 H
mijllll]llll|llll| )0 110.0 120.0
Fig. 5. TLC pattern of 99mTc-AEDP complex in 0.9% NaCl. In vivo experiments
Experiments in rats were carried out in accordance with appropriate European Community directive guidelines (86/609/EEC). Biodistribution studies were performed in healthy Wister Han rats (male, 160-220 g). 0.3 ml of 99mTc-AEDP, 99mTc-HEDP or 99mTc-MDP (2.2-3.7 MBq) in saline were administered to rats intravenously via the tail vein. The animals were anesthetized and sacrificed, routinely 1, 3, and 24 h post-intravenous injection (p.i.), and selected organs were taken out. The radioactivity of weighted samples of femur, heart, liver, lungs, kidneys, spleen, stomach, intestine, and blood were measured using a gamma counter CE/SN:03 L 504. The uptakes in the different selected organs, expressed as %ID(±SD)/g, of the organs for all the radiolabeled phosphonate complexes are given in Tables 1-3. Bone-to-blood and bone-to-muscle uptake ratios were determined from the %ID/g values for the organs.
Imaging studies were performed in normal rats (Wistar Han, male, 160-220 g) at 1 h after intravenous injection of 29.6 MBq (0.8 mCi) of 99mTc-AEDP, 99mTc-HEDP or 99mTc-MDP, respectively.
Results and discussion
New product AEDP was synthesized according to the reaction scheme shown in Scheme 1. The synthesis
was started with the addition of phosphoryltrichlo-ride to a mixture of acetonitrile and phosphorous acid to obtain crude AEDP. The solid product was separated by filtration, then washed with water and methanol to give pure AEDP in 77% yield.
AEDP was characterized by 13C, 31P, JH-NMR, XRD, EA, and IR spectroscopic techniques. JH-NMR spectrum of AEDP showed a triplet peak representing the phosphorus splitting of the methyl hydrogens.
AEDP was labeled with 99mTc in high labeling yields. Radiochemical purity and radiochemical yields were determined by ITLC and found to be greater than 97% (Fig. 4).
Biodistribution of 99mTc-AEDP was studied in rats and the results were compared with those obtained using 99mTc-HEDP and 99mTc-MDP. One hour post injection, 99mTc-AEDP, 99mTc-HEDP, and 99mTc-MDP showed significant uptake by bone. Femur was taken as a representative of the skeleton and observed uptakes in femur were 5.54%/g, 12.7%/g, and 4.67%/g for 99mTc-AEDP, 99mTc-HEDP, and 99mTc-MDP, respectively, at 1 h post injection. No leaching of the activity from bone was observed for 99mTc-MDP from 1 to 3 h post injection. In contrast, the activity of bone was decreased after 24 h post injection for 99mTc-AEDP. Also, the activity of bone was decreased after 3 h for 99mTc-HEDP. No increase of the uptake in any of the organs and tissues were observed with exception in stomach for 99mTc-AEDP. The activities in blood were 1.38%/g, 3.65%/g, and 1.7%/gfor 99mTc-AEDP, 99mTc-HEDP, and 99mTc-MDP, respectively, at this time point and no significant accumulation of the activity was observed in any of the major organs except in kidneys. However, the uptake observed in kidneys showed an increase up to 3 h, then decreased from 3 to 24 h for both 99mTc-AEDP and 99mTc-HEDP. In contrast, the uptake was decreased from 1 to 3 h, then increased from 3 to 24 h for 99mTc-MDP. From 30 to 50% of the injected activity was cleared via urinary excretion within 3 h post injection for all the complexes. The bone-to-muscle uptake ratios of 99mTc-AEDP were 167 and 242 at 3 and 24 h, respectively. While the bone-to-muscle uptake ratios of 99mTc-HEDP and 99mTc-MDP were 21.4, 367 and 95.6, 211 at 3 and 24 h, respectively, as given in Tables 1-3.
Table 1. Biodistribution pattern of 99mTc-AEDP complex in Wistar rats. AEDP: (1-aminoethane-1,1-diyl)diphosphonic acid
Biodistribution of 99mTc-AEDP [%ID/g]
Blood 1.38 ± 0.81 0.51 ± 0.17 0.37 ± 0.06
Liver 0.13 ± 0.06 0.07 ± 0.017 0.44 ± 0.02
Intestine 0.17 ± 0.002 0.50 ± 0.08 0.25 ± 0.07
Kidney 2.00 ± 0.59 3.17 ± 0.28 2.60 ± 0.17
Stomach 0.14 ± 0.003 0.21 ± 0.13 0.36 ± 0.24
Heart 0.15 ± 0.08 0.04 ± 0.01 0.03 ± 0.012
Lungs 0.25 ± 0.08 0.11 ± 0.019 0.06 ± 0.007
Femur 5.54 ± 0.98 5.01 ± 0.76 2.42 ± 0.31
Spleen 0.14 ± 0.01 0.07 ± 0.01 0.45 ± 0.09
Muscle 0.04 ± 0.001 0.03 ± 0.001 0.01 ± 0.001
Femur/blood 4.01 9.8 6.54
Femur/muscle 138 167 242
Table 2. Biodistribution pattern of 99mTc-HEDP complex in Wistar rats. HEDP: (1-hydroxyethane-1,1-diyl)diphos-phonic acid
Biodistribution of 99mTc-HEDP [%ID/g]
Organ 1 h 3 h 24 h
Blood 3.65 ± 2.05 0.74 ± 0.61 0.04 ± 0.01
Liver 0.14 ± 0.03 0.07 ± 0.02 0.01 ± 0.003
Intestine 0.18 ± 0.03 0.13 ± 0.03 0.03 ± 0.009
Kidney 1.53 ± 0.05 1.67 ± 1.20 0.52 ± 0.134
Stomach 0.07 ± 0.004 0.28 ± 0.08 0.06 ± 0.10
Heart 0.20 ± 0.03 0.04 ± 0.009 0.005 ± 0.002
Lungs 0.28 ± 0.04 0.14 ± 0.009 0.01 ± 0.002
Femur 12.71 ± 4.50 4.93 ± 0.22 3.67 ± 0.43
Spleen 0.13 ± 0.02 0.09 ± 0.02 0.03 ± 0.002
Muscle 0.28 ± 0.02 0.23 ± 0.02 0.01 ± 0.001
Femur/blood 3.48 6.5 91.75
Femur/muscle 45 21 367
Table 3. Biodistribution pattern of 99mTc-MDP complex in Wistar rats. MDP: methylenediphosphonic acid
Organ Biodistribution of 99mTc-MDP [%ID/g]
1 h 3 h 24 h
Blood 1.70 ± 0.6 0.29 ± 0.023 0.23 ± 0.12
Liver 0.22 ± 0.31 0.05 ± 0.02 0.48 ± 0.15
Intestine 0.31 ± 0.007 0.21 ± 0.16 0.15 ± 0.07
Kidney 1.11 ± 0.83 0.97 ± 0.02 1.86 ± 0.18
Stomach 0.44 ± 0.11 0.22 ± 0.30 0.08 ± 0.04
Heart 0.12 ± 0.04 0.12 ± 0.021 0.029 ± 0.01
Lungs 0.20 ± 0.004 0.13 ± 0.03 0.09 ± 0.016
Femur 4.67 ± 0.03 6.69 ± 0.34 4.22 ± 0.71
Spleen 0.11 ± 0.01 0.08 ± 0.02 1.08 ± 0.19
Muscle 0.08 ± 0.007 0.07 ± 0.006 0.02 ± 0.001
Femur/blood 2.74 23 18.34
Femur/muscle 58.8 95.6 211
Scintigraphic images of 99mTc-AEDP, in normal rats revealed highly selective skeletal uptake compared with both 99mTc-HEDP and 99mTc-MDP (Fig. 6).
Conclusion
A new bisphosphonate analogue as ligand for bone study was developed and evaluated in rats. The labeling efficiency of 99mTc-AEDP, as determined by ITLC, was greater than 97%. Radioactivity in bone tissue was as high as 5.54 ± 0.98% ID/g (mean ±
SD, n = 6) at 1 h after injection and decreased to 2.42 ± 0.31% ID/g at 24 h. Activity in kidneys was the highest at 3 h after injection. The radioactivities in muscle, stomach, small intestine, liver, and blood were all lower than 0.6% ID/g from 1 to 24 h.
The bone-to-blood uptake ratio was 4.01 at 1 h and increased to 9.8 at 3 h post injection, then decreased to 6.54 at 24 h. While, the bone-to-muscle uptake ratio increased from 138 at 1 h to 242 at 24 h. A major part of the injected radiotracer (~54%) was excreted by the urinary system by 3 h.
Fig. 6. Whole-body image of rat corresponding to 1 h after injection of 0.8 mCi (29.6 MBq) of 99mTc-AEDP, 99mTc-HEDP and 99mTc-MDP. AEDP: (1-aminoethane-1,1-diyl)diphosphonic acid; MDP: methylenediphosphonic acid; HEDP: (1-hydroxyethane-1,1-diyl)diphosphonic acid.
In conclusion, 99mTc-AEDP appears to be a good potential candidate for clinical use as a bone-seeking agent, since it displays highly selective uptake in the skeletal system, has low non-target uptake, and rapid clearance in blood. Scintigraphic images of 99mTc-AEDP in normal rats revealed selective skeletal uptake.
Acknowledgments. The author would like to thank both Professor I. Othman (DG of AECS) for the encouragement and support and Professor A. H. Al--Rayyes, head of the Radioisotope Department, for his support of this work. Thanks to Dr. M. Zein at Al Assad Damascus University hospital for performing SPECT scan. We also thank N. Karajoli, R. Ajaya, N. Alwan, T. Saleh, and I. Kulaisheh for their kind assistance with the laboratory work.
References
1. Jurisson, S., & Lydon, S. (1999). Potential technetium small molecule radiopharmaceuticals. Chem. Rev., 99(9), 2205-2218. DOI: 10.1021/cr980435t.
2. Fleisch, H. A. (1998). Bisphosphonates: mechanisms of action. Endocr. Rev., 19(1), 80-100.
3. Papapoulos, S. E. (1995). Pharmacodynamics of bisphosphonates in man. In O. Bijvoet, H. Fleisch, R.
E. Cornfield, & G. G. Russell (Eds.), Bisphosphates on bone (Chapter 15). Amsterdam: Elsevier Science B.V
4. Subramanian, G., McAfee, J. G., Blair, R. J., Kallfelz,
F. A., & Thomas, F. D. J. (1975). Technetium-99m--methylene diphosphonate - a superior agent for skeletal imaging: comparison with other technetium complexes. J. Nucl. Med, 16(8), 744-755. DOI: 10.2174/092986712801215865.
5. Bevan, J. A., Tofe, A. J., Benedict, J. J., Francis, M. D., & Barnett, B. L. (1980). Tc-99m HMDP (Hydroxymeth-yleneDiphosphonate): A radiopharmaceutical for skeletal and acute myocardial infarct imaging. I. Synthesis and distribution in animals. J. Nucl. Med., 21(10), 961-966. DOI: 10.2174/092986712801215865.
6. Bartholoma, M. D., Louie, A. S., Valliant, J. F., & Zubieta, J. (2010). Technetium and gallium derived radiopharmaceuticals: comparing and contrasting
the chemistry of two important radiometals for the molecular imaging era. J. Chem. Rev., 110(5), 2903-2920. DOI: 10.1021/cr1000755.
7. Russell, R. G. G., & Fleisch, H. (1975). Pyrophosphate and diphosphonates in skeletal metabolism. Clin. Orthop., 108, 241-263.
8. Shinoda, H., Adamek, G., Felix, R., Fleisch, H., Schenk, R., & Hagan, P. (1983). Structure-activity relationships of various bisphosphonates. Calcif. Tissue Int., 35, 87-99. DOI: 10.1007/BF02405012.
9. Schenk, R., Eggli, P., Fleisch, H., & Rosini, S. (1986). Quantitative morphometric evaluation of the inhibitory activity of new. Amino bisphosphonates on bone resorption in the rat. Calcif. Tissue Int., 38, 342-349. DOI: 10.1007/BF02555748.
10. Pluijm, G. V D., Binderup, L., Bramm, E., Wee-Pals, L. V D., Groot, H. D., & Binderup, E. (1992). Disodium 1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1--bisphosphonate (EB-1053) is a potent inhibitor of bone resorption in vitro and in vivo. J. Bone Miner. Res., 7, 981-986. DOI: 10.1002/jbmr.5650070815.
11. Sato, M., & Grasser, W (1990). Effects of bisphos-phonates on isolated rat osteoclasts as examined by reflected light microscopy. J. Bone Miner Res., 5, 31-40. DOI: 10.1002/jbmr.5650050107.
12. Rudd, T. G., Allen, D. R., & Hartnett, D. E. (1977). Tc-99m methylene diphosphonate versus Tc-99m pyrophosphate: biologic and clinical comparison. J. Nucl. Med., 18, 872-876.
13. Lin, W. Y., & Wang, S. J. (1996). The influence of two bone agents (Tc-99m pyrophosphate and Tc-99m meth-ylenediphosphonate) on quantitative sacroiliac joint scintigraphy. Nucl. Med. Commun., 17, 1035-1038.
14. Imanishi, Y., Mitogawa, Y., Takizawa, M., Konno, S., Samura, H., & Ohsawa, A. (1999). Relapsing polychondritis diagnosed by Tc-99m MDP bone scin-tigraphy. Clin. Nucl. Med., 24, 511-513.
15. Altomare, A., Cuocci, C., Giacovazzo, C., Mo-literni, A., & Rizzi, R. (2008). A computer program for qualitative analysis using powder diffraction data. J. Appl. Cryst, 41, 815-817. DOI: 10.1107/ S0021889808016956.
16. Tsaryk, N. V, Dudko, A. V, Kozachkova, A. N., & Pekhnyo, V I. (2011). A second triclinic polymorph of (1-ammonio-1-phosphonoethyl)phosphonate. Acta Crystallogr. Sect. E-Struc. Rep. Online, 67, o1651-o1652. DOI: 10.1107/S1600536811022239.
