MIBGHS standard reference material was purchased from European Pharmacopeia (EP). Analytical reference materials including m-bromobenzylguanidine (MBrBG), m-bromobenzylamie (MBrBA), and m-iodobenzylamine (MIBA) hydrochloride were purchased from Abcr, GmbH (Germany), Alfa Aesar (USA) and Sigma-Aldrich (USA), respectively. Several different batches of MIBGHS were synthesized and purified by the Chemistry Division at the INER and stored at −18˚C - −20˚C until analysis. The HPLC analytical columns for analysis of MIBG were reversed-phase C8 (150 × 4.6 mm, particle size 5 μm, Agilent Eclipse XBD) and phenyl-type (100 × 2.1 mm, particle size 3 μm, Hypersil Gold phenyl, Thermo Fisher, USA).

To identify the impurities in MIBG, determining suitable mass spectrometer parameters and fragmentation patterns for MIBG is the first issue. A solution of MIBG (EP standard reference material) in aqueous ammonium formate (5 mM) and acetonitrile (1:1) was supplied to the 4000QTRAP. The intensity was optimized at DP 10 - 30 V, curtain gas 10 - 30 psi, IS 4800 - 5200 V, CE 20 - 60 V, EP 10 - 30 V, and GS1, GS2 both 10 psi.

The m/z values of the moleculr ions of MIBG are 276 and 277 and those of the fragmentation product ions of MIBG (m/z 276) were 259, 234, 217, 149, and 90, which indicated the fragmentation scheme of MIBG outlined in Figure 2. These results indicate that the weak chemical bonds in MIBG include the phenyl-iodide and C-N bonds, because the parent ion possesses I·, NH₃, cyanamide, and guanidinium leaving groups (Δm/z = −127, −17, −42, and −59, respectively) that results in product ions with more delocalized and stable structures. An interesting point here is that when the iodo-radical leaves from

phenyl group, a transient seven-member aromatic conjugated radical cation is formed.

Analytical methods for determining the purity of MIBG are reported in the U.S. Pharmacopeia (USP) [23], and ¹³¹I-MIBG metabolites in the plasma of patients with metastatic pheochromocytoma are separated on a reversed phase C18 column [24] [25] and detected by UV absorbance. In those studies, the mobile phase contained phosphoric acd and TEA as ion pair agents because of the high polarity and positive charge of the guanidinium group. However, phosphate and TEA suppress electrospray ionization efficiency and reduce mass spectra signals [26]. No HPLC-MS/MS method for MIBG and its derivatives has been reported yet. Consequently, we initially tried to separate the impurity in our lab-synthesized MIBG on a C18 column with an isocratic mobile phase comprising a phosphoric acid-TEA aqueous buffer and acetonitrile (9:1) (USP method), but no contaminants were observed in the chromatogram (Figure 3) by UV detection.

Thus, we initially employed an HPLC method involving a C8 column and phosphate, TEA mobile phase to determine MIBG purity and allow tandem MS analysis, but we also later developed a method without phosphate and TEA using a phenyl column to determine the MIBG impurities and derivatives by mass spectrometry.

The starting materials used in our MIBG synthesis included m-iodobenzylamine hydrochloride (MIBA. HCl), cyanamide (N≡C‒NH₂), and potassium bicarbonate, which were refluxed, and the product was then precipitated and recrystallized as the hemisulfate salt. The purities of MIBGHS over different preparative batches were all above 99.0% according to analysis using the C8 column and isocratic elution. A major impurity peak (0.3% - 0.5%) was observed with molecular ions of m/z 230 and 228 (isotope distribution ratio ca. 1:1, indicating that it contains Br). The fragmentation ion mass spectra of the impurity showed signal for 230 à 213, 188, 171, and 90; and 228 à 211, 186, 169, and 90. Therefore the impurity was determined to be the bromo-substituted analog, MBrBG (Figure 4), where the iodo of MIBG is substituted with a bromo. Therefore, the MIBA starting materal was analyzed by HPLC (C8 column), and the chromatogram showed a peak area at retention time (t_R) 6.1 min of 99.8% and a trace impurity (t_R 4.1 min, 0.2%). The m/z values of the impurity molecular ion were 188 and 186, and those of their fragmentation product ions pairs were 171, 90 and 169, 90 respectively (Figure 5). Therefore the impurity in MIBA was MBrBA. The HPLC and MS results were also confirmed using purchased reference materials, MBrBA and MBrBG. Based on our conclusions, it is indicated that the starting material MIBA should be analyzed for impurity before the first time it is employed to prepare MIBG. A new batch (#0000000624, replacing the previously used #101582951) of the MIBA. HCl agent was employed

to prepare the later batches of MIBG, and neither the MIBG product nor the MIBA starting material contained bromo-sunstituted analogs.

In order to improve chromatographic efficiency and thus analyze more complex derivatives of MIBG, an HPLC-MS/MS method without TEA, TFA and phosphoric acid in the mobile phase, which interfere with mass spectrometry analysis, was developed. In this method, a reversed-phase column with phenyl groups (Hypersil Gold phenyl column) was employed, as π-π interaction between analytes and stationary phase enhances the separation efficiency of MIBG [27]. Figure 6 shows the resultant chromatogram of MIBG (purity > 99.4%) with the number of theoretical plates (N) at 10,000 and a retention time of 4.8 min. The peak is sharp and thus very easily distinguished from impurities. Consequently, this chromatographic method was later applied to study the stability and degradation products/ causes.

The short-term stability of MIBG strored at 25˚C ± 3˚C under a relative humidity of less than 75% in the dark for 1, 2, 3, 6 months, and the long-term stability of MIBG stored at −18˚C ± 2˚C, in the dark for 3, 6, 9 and 12 months were evaluated. The chromatograms of MIBG were steady (>99%) without observable degeneration over 6 months and 12 months, respectively. Thus, MIBGHS is stable for at least six and twelve months under room temperature and frozen storage conditions. There were two major impurities constantly

existed since initial MIBGHS (termed Imp 1 and 2) but the amount didn’t increase over storage periods. The retention time, molecular ion mass (MS¹), and peak aera % for the impurities were 7.7 min, 532, 0.1% - 0.2% and 7.95 min, 492, 0.25% - 0.35%, respectively. (The MS¹ values for the impurities were confirmed by extraction ion count (XIC) to be 532 - 533 and 492 - 493 in LC-MS chromatogram of MIBG).

The identities of the impurities were determined by fragmentation structures based on the MS/MS spectra. (Figure 7(a), Figure 7(b)). Imp 1, N³,N⁵-bis(3-iodobenzyl)-4H-1,2,4-triazole-3,5-diamine, is a dimer of two MIBG molecules coupled through condensation of the guanidine groups, where an ammonia molecule and tow protons are eliminated to form a five-memberd ring, i.e., a triazole-diamine compound. Imp 2, 1,3-bis(3-iodobenzyl)guanidine, is the coupling product of MIBG with MIBA via a condensation reaction that eliminates an ammonia molecule. Furthermore, there were several trace impurities shown in the MIBG chromatogram with peak areas < 0.1%, but their mass spectra (MS¹) were covered by background noise. Hence, an MIBG solution was perfused into the mass spectrometer inorder to identify more contaminants.

Contaminants with MS¹ values of m/z 494, 534, 551, 649, and 671 were found and their enhanced product ion (EPI) scans all contained signals for m/z 276, 259, 234, 217, and 90. Furthermore, the precursor ion scan for m/z 276 including 534, 551, and 649 shows that they are MIBG adducts. The identities of the contaminants with MS¹ 534 and 494 may possibly be di(3-iodobenzyl)guanidinylamine (MS² 534 à 517, 492, 475, 450, 276, etc.) and N,N’-bis(3-iodobenzyl)

methanetriamine (MS² 494 à 477, 452, 434, 276, etc.), respectively (Figure 8(a), Figure 8(b)). Both are analogs of Imp 1 and 2. The contaminants with MS¹ 551, 649, and 671 are MIBG₂·H⁺, MIBG₂∙H₂SO₄·H⁺, and MIBG₂·H₂SO₄·Na⁺, respectively. Thus these three molecular ions should be seen as representing MIBG itself instead of its derivatives. It is believed that contaminants Imp 1 and 2 and two other analogs in MIBGHS come from byproducts formed during synthesis due to guanidine or amino group overreaction and are not formed during storage.

The potential derivatives of MIBGHS upon improper storage and transport or reprocess were investigated by forced degradation under harsh conditions, including acidic/alkaline, oxidative, thermal, and UV photolysis factors. The chromatograms of MIBG incubated under acidic condition (HCl 3%) show that it is stable over 72 h without spoilage. This is reasonable as MIBG is recrystalizated as the hemisulfate salt after adding diluted H₂SO₄. For alkaline condition (TEA_(aq), 2%), the purity remains at > 98% for the first 24 h, but falls to 90% by 72 h. Two degradation products are detected in the chromatogram (DP_b1, t_R = 3.97 min, 5.4% and DP_b2, t_R = 5.26 min, 3.5%). Their MS/MS data are DP_b1: 234 à 217, and DP_b2: 346 à 328, 302, 276, 260, 232, and 217. The mass spectra and fragmentation structures of DP_b2 are shown in Figure 9. Therefore, DP_b1 and DP_b2 were determined to be m-iodobenzylamine (MIBA) and 4-hydroxy-1,3-diazet-2-yl(3-iodobenzyl)carbamate.

After exposure to oxidative surrounding for 24 h, an oxidized product (DP_ox1, t_R = 5.0 min, 1%) with MS¹ 292 (ΔMS = +16 cf. MIBG) and fragment ion masses of 275, 250, 233, and 217. The mass spectra and fragment structures of DP_ox1 are shown in Figure 10. It is supposed that oxidization occurs on the iodide to form a hydroxyiodonium species, which is similar to the oxidization behavior of epidepride [28].

For MIBGHS powder stayed in a thermal environment (80˚C for 72 h), MIBG does not spoil and no derivatives are produced. However, in aqueous solution, the purity falls to 96.9% (24 h) and one derivative is produced, i.e., DP_th1, which has m/z = 234 and MS² = 217. Thus, DP_th1 is MIBA (2.5%). This indicates that MIBG may decompose a little during the radioiodine substitution reaction at 175˚C (provide activation energy to induce isotope replacement) for 1 h, although thermal decomposition might result in MIBG losing cyanamide, especially promoted by the aqueous environment. However, MIBGHS powder is inert to thermal effects. Trace MIBA may contaminate the radiolabeled product *I-MIBG and reduce its effective dose.

When the MIBG solution is exposed to UV light for 22 and 42 h, the amount of MIBG decreases to 95% and 91%, respectively. Three decomposition products are observed in the chromatograms, and their fragmentation mass spectra revealed the transitions DP_UV1: 150 à 133, 108, 91 (benzylguanidine, t_R = 1.1 min), DP_UV2: 166 à 149, 131, 124, 117, 107, 103 (hydroxybenzylguanidine), and

DP_UV3: 164 à 147, 135, 122, 120, 119 [(hydroxybenzylidene)guanidine)]. DP_UV2 and DP_UV3 co-eluate at t_R = 1.4 min. However, the MIBGHS powder is more stable under UV irradiation (>97% till 42 h) than the aqueous solution, with only DP_UV1 being produced. The result indicates the weakness of the iodo-phenyl bond, which is partially broken under UV irradiation, then hydroxide filling the resultant vacancy. The mass spectra and fragment structures of DP_UV1 - 3 are shown in Figures 11(a)-(c).