{"id":56101,"date":"2024-03-20T10:22:02","date_gmt":"2024-03-20T10:22:02","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=56101"},"modified":"2024-04-02T04:27:00","modified_gmt":"2024-04-02T04:27:00","slug":"stress-testing-of-pidotimod-by-lc-and-lc-ms-ms","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no1\/stress-testing-of-pidotimod-by-lc-and-lc-ms-ms\/","title":{"rendered":"Stress Testing of Pidotimod by LC and LC-MS\/MS"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Poli Industria Chimica, an Italian enterprise, discovered Pidotimod (PDM) and launched it for commercial use in 1993. PDM was the first peptide-like, biological response modifier that activates both primary and acquired immune responses to bacteria and viruses1,2<\/sup>. PDM has been studied in vitro<\/em> and in vivo<\/em>3<\/sup> for prevention, management and treatment of acute ARTIs (respiratory tract infections) in children. Although most of the studies on PDM were published over 25 years ago, it is still utilized to prevent ARTIs today. Despite the fact that publications were available during the approval of PDM, there is a void in the literature in terms of complete pharmacological and analytical research. PDM research has recently looked into new ways for inducing primary and secondary immune responses4-6<\/sup>. PDM was also reevaluated7<\/sup> after a few years, and a new synthetic method was published8<\/sup>.<\/p>\n\n\n\n

An exhaustive literature search found that only few analytical procedures for estimating PDM have been established and reported, including HPLC-UV9<\/sup>; HILIC-MS\/MS12<\/sup>; HPLC\u2013MS10<\/sup>; and HPLC-MS\/MS11<\/sup>. A bioequivalence study of two formulations of PDM was published13<\/sup>. There is only one published study on the use of GC to determine residual organic solvents in PDM14<\/sup>. Crimella et al.15<\/sup> described the synthesis and pharmacological evaluation of  PDM; it\u2019s carboxamido derivatives, enantiomers and diastereomers16<\/sup>, as well as the analytical and chemical profile of PDM in 1994. They found two process-associated impurities, as well as two other impurities not connected to the process, in an industrial batch of PDM. On the CHIRALPAK17<\/sup> and CHIRADEX18<\/sup> stationary phases, chiral separation of PDM and its enantiomers has been described. PDM enantiomers have recently been studied in terms of thermodynamic characteristics, crystal structure, and molecular docking19<\/sup>.<\/p>\n\n\n\n

Despite its widespread use and effectiveness in mitigating ARTIs, only one study on PDM impurity profiling was published15<\/sup>. To our knowledge, stress degradation and structural characterization of PDM degradation products (DPs) have not been published in the literature. As a result, the aim of the work was to investigate Pidotimod degradation behaviour under ICH-recommended stress degradation parameters. To separate diverse degradation products, a liquid chromatographic technique was developed. The structure of degradation products were speculated, and fragmentation pathways were described, based on an LC-MS-MS investigation.<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Reagents and Chemicals <\/strong><\/p>\n\n\n\n

Swapnaroop\npharmaceuticals in Aurangabad, India, provided the PDM bulk drug. Spectrochem .\nLimited Mumbai, India; supplied HPLC graded ammonium acetate; MeOH (methanol)\nand ACN (acetonitrile). Unless otherwise specified,\nthe solutions were filtered via nylon 6, 6, membrane filter [0.2 \u00b5; Ultipor\u00ae\nN66\u00ae from Pall Life Sciences Limited; USA] before use. SD Fine chemicals; Mumbai,\nIndia, supplied analytical grade HCl (hydrochloric acid); NaOH (sodium\nhydroxide); and H2<\/sub>O2<\/sub> (hydrogen peroxide).<\/p>\n\n\n\n

Chromatographic Conditions and Equipment\u2019s<\/strong><\/p>\n\n\n\n

Thermal\nlining services, Vadodara, India, provided precision water baths with\ntemperature controllers for degradation experiments. In a photo-stability\nchamber (by Thermolab Scientific Equipments; Limited, Vadodara) coupled by a\nlight pool containing fluorescent (OSRAM L20) and 4 UV (OS-RAM L73) lamps,\nphotolytic degradation was studied. Thermal and humidity chamber (by SR Labs\nInstruments; Maharastra) was utilized for the evaluation of temperature –  humidity conditions.<\/p>\n\n\n\n

Waters Acquity from Waters Corporation: Milford: USA; with Empower 2 software and PDA detector was used for HPLC analysis. At wavelength of 215 nm, the separation was carried out using RP C18 column (Phenomenex; 5 \u00b5m; 250 mm and 4.6 mm,). The mobile phase was made up of  97:03 v\/v mixture of buffer (ammonium acetate;10 mM, with pH 4.5 balanced with glacial acetic acid) and 90:10 v\/v of MeOH\/CAN, at  flow rate of 1.0 ml\/min with isocratic elution. The experimentation was carried out in a column oven at 400<\/sup>C with a 20-\u00b5L injection volume. “The LC-MS-MS experiments were run in positive and negative ESI mode utilizing Xcalibur software on the LCQ fleet, a Thermo Fisher scientific instrument connected with an Agilent HPLC 1100 series quaternary system delivery module.” Following parameters were set for experimentation: nebulizer pressure =20 psi, gas temperature = 2500<\/sup>C (using nitrogen as a drying gas at pressure of 30 psi) and capillary voltage = 5500 V”20<\/sup>. <\/p>\n\n\n\n

Preparation\nof Degradation Products (DPs)<\/strong><\/p>\n\n\n\n

Various\nstress conditions were applied for the bulk drug\nin the stress degradation experiments. Placebo samples were developed to\ncompare with the stress-degraded samples.<\/p>\n\n\n\n

10 mL of 1 mg\/mL of PDM was prepared in\n0.8 N HCl or 0.1 N NaOH and was refluxed (800<\/sup>C; in dark) for 3\nhours, for acid and base degradation. Before analysis, a 2 mL aliquot of the\nsamples was reserved, neutralized using the same strength of NaOH or HCl, and\nstored at -100<\/sup>C. Sample of 10 mL of 1 mg\/mL was prepared in double distilled water and refluxed (800<\/sup>C;\nin  dark) for 6 hours for neutral\ndegradation. Oxidative degradation was carried out in 10 mL of 1 mg\/mL of PDM that\nwas produced with 0.01 percent H2<\/sub>O2<\/sub> and refluxed at 800<\/sup>C\nin the dark for 1.5 hours. API (Active Pharmaceutical\nIngredient) was smeared 1mm thick on a petri dish and exposed to UV\nlight (5382 LUX and 144 UW\/cm2<\/sup>; for 21 days) for photolytic\ndegradation. API was spreaded in 1mm thickness in petridish and was placed in\nan oven (800<\/sup>C; for 21 days) in the dark to prepare the thermal degradation\nproduct. For thermal humidity degradation, PDM was kept in the stability\nchamber for 21 days (40\u00baC\u00b12\u00baC; 75\u00b15 percent RH).<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

Method Development <\/strong><\/p>\n\n\n\n

To segregate the peaks of PDM and DPs, the DPs generated under the stress conditions were combined and diluted to 150 ppm with respect to PDM. PDM is polar in nature due to its dipeptide structure. Because DPs (excluding DP4) were even more polar than PDM, a greater buffer ratio was used to lengthen retention time (Rt) and resolve the DPs. The pH of the buffer had a significant impact on PDM Rt. Lowering the buffer pH to 3.8 delayed PDM Rt and resolved DP3, DP2, and DP1. The Rt of PDM reduced when the pH was raised to 5.8, and DP3, DP2, and DP1 were co-eluted. At all pH levels, DP4 was well resolved. Despite the fact that ACN has a higher sensitivity, it gives shorter Rt of PDM. DP3, DP2, and DP1 were also co-eluted, making resolution challenging at larger buffer ratios. MeOH increases the Rt of PDM and helps to resolve DPs at greater buffer ratios, but it decreases sensitivity. To optimize Rt, resolution, and sensitivity, a combination of MeOH and ACN (90:10 v\/v) was used. Rt, peak symmetry, and sensitivity of PDM and DPs are all affected by column temperature. As a result, optimization required the use of ammonium acetate buffer and MeOH\/ACN. The best separation was accomplished with a mobile phase consisting buffer (ammonium acetate; pH 4.5, 10 mM) and MeOH\/ACN (90:10 v\/v) in a ratio of 97:03 v\/v at 400<\/sup>C. Stressed samples were scanned in a PDA detector (200 to 400 nm); PDM and DPs exhibited good sensitivity at 215 nm, therefore detection was carried out at this wavelength. Figure 1a shows the final chromatogram developed with the chosen mobile phase, which shows highly resolved peaks of PDM and DPs. <\/p>\n\n\n\n

Stress Degradation studies<\/strong><\/p>\n\n\n\n

When degradant mixture was evaluated in LC PDA, peaks of five DPs were obtained, as shown in figure 1a. Table 1 summarizes the forced degradation settings with percent degradation (percent Deg) under various situations. The formula for calculating percent Deg21<\/sup> used was-<\/p>\n\n\n\n

PDM\nwas the most vulnerable to oxidative deterioration. Under acid and basic\ndegradation, it degrades significantly. Photolytic, thermal humidity and\nneutral degradation showed minor deterioration. The drug is most stable under\nthermal degradation. Figure 1b shows the chromatograms of individual stressed\nsamples. <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: a) Optimized chromatogram, b) Individual chromatograms of various degradation conditions<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 1: PDM and synthetic mixture stress degradation summary<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Stressor<\/strong><\/p>\n<\/td>\n

\n

Conc. of Stressor<\/strong><\/p>\n<\/td>\n

\n

Time<\/strong><\/p>\n<\/td>\n

\n

% Deg (API)<\/strong><\/p>\n<\/td>\n

\n

% Deg (synthetic mixture)<\/strong><\/p>\n<\/td>\n

\n

DPs formed with Rt<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Acid degradation<\/p>\n<\/td>\n

\n

0.8 N, 800<\/sup>C<\/p>\n<\/td>\n

\n

3 Hrs<\/p>\n<\/td>\n

\n

 <\/p>\n

46.44<\/p>\n<\/td>\n

\n

 <\/p>\n

46.21<\/p>\n<\/td>\n

\n

DP1(2.88),<\/p>\n

DP2(3.40),<\/p>\n

DP3(3.74),<\/p>\n

DP4(12.22)<\/p>\n<\/td>\n<\/tr>\n

\n

Base degradation<\/p>\n<\/td>\n

\n

0.1 N, 800<\/sup>C<\/p>\n<\/td>\n

\n

3 Hrs<\/p>\n<\/td>\n

\n

 <\/p>\n

 <\/p>\n

61.25<\/p>\n<\/td>\n

\n

 <\/p>\n

 <\/p>\n

60.99<\/p>\n<\/td>\n

\n

DP1(3.01),<\/p>\n

DP2(3.43),<\/p>\n

DP3(3.82),<\/p>\n

DP4(12.86)<\/p>\n<\/td>\n<\/tr>\n

\n

Neutral degradation<\/p>\n<\/td>\n

\n

800<\/sup>C<\/p>\n<\/td>\n

\n

6 Hrs<\/p>\n<\/td>\n

\n

10.4<\/p>\n<\/td>\n

\n

10.1<\/p>\n<\/td>\n

\n

DP1(2.91),<\/p>\n

DP3(2.84),<\/p>\n

DP4(12.22)<\/p>\n<\/td>\n<\/tr>\n

\n

Oxidative degradation<\/p>\n<\/td>\n

\n

0.01 %, 800<\/sup>C<\/p>\n<\/td>\n

\n

1.5 Hrs<\/p>\n<\/td>\n

\n

75.38<\/p>\n<\/td>\n

\n

74.14<\/p>\n<\/td>\n

\n

DP1(2.94),<\/p>\n

DP2(3.24),<\/p>\n

DP3(3.79),<\/p>\n

DP5(6.19)<\/p>\n<\/td>\n<\/tr>\n

\n

Photolytic degradation<\/p>\n<\/td>\n

\n

—<\/p>\n<\/td>\n

\n

21 days<\/p>\n<\/td>\n

\n

8.25<\/p>\n<\/td>\n

\n

8.0<\/p>\n<\/td>\n

\n

DP1(2.90),<\/p>\n

DP2(3.28),<\/p>\n

DP3(3.86),<\/p>\n

DP4(12.36)<\/p>\n<\/td>\n<\/tr>\n

\n

Thermal degradation<\/p>\n<\/td>\n

\n

800<\/sup>C<\/p>\n<\/td>\n

\n

21 days<\/p>\n<\/td>\n

\n

No degradation<\/p>\n<\/td>\n

\n

No degradation<\/p>\n<\/td>\n

\n

No degradation<\/p>\n<\/td>\n<\/tr>\n

\n

Thermal Humidity induced degradation<\/p>\n<\/td>\n

\n

400<\/sup>C 20<\/sup>C and 750<\/sup>C 50<\/sup>C<\/p>\n<\/td>\n

\n

21 days<\/p>\n<\/td>\n

\n

 <\/p>\n

5.2<\/p>\n<\/td>\n

\n

 <\/p>\n

5.0<\/p>\n<\/td>\n

\n

DP1(2.91),<\/p>\n

DP3(3.84),<\/p>\n

DP4(11.98)<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

Structural characterization of PDM and DPs<\/strong>\n<\/em><\/strong><\/p>\n\n\n\n

MS-MS Spectra of PDM (m\/z 245)<\/strong><\/strong><\/p>\n\n\n\n

PDM’s [M+H]+<\/sup> ion’s ESI-MS\/MS\nspectrum revealed that the loss of water resulted in the most prevalent\nfragment ion at m\/z 227. Fragmentations of INH-2 (Inherent impurity-2) i. e.\nthiazolidine carboxylic acid [15], gives fragment ion at m\/z 88 as a result of\nloss of \u2013COO+H2<\/sub>. The spectra also revealed a low abundance fragment ion\nwith an m\/z of 187.<\/p>\n\n\n\n

MS-MS Spectra of DP\u2019s<\/strong><\/p>\n\n\n\n

As\nstated in table 2, PDM has been shown to have four impurities[15]<\/sup>.Two\nprocess related impurities (INH-1 and INH-2) were observed at Rt of 2.95 and\n3.79. These processes related impurities were also formed during stress degradation\nand were identified as DP1 and DP3 respectively. The MW of INH-1 was determined\nto be 129 after an LC-MS-MS analysis, which corresponds to the process impurity\nPyroglutamic acid; whereas the MW of INH-2 was found to be 133, which\ncorresponds to the process impurity thiazolidine carboxylic acid. Also MW of\nDP-4 (MW 244) matched with the impurity reported by Crimella T [15]. Other DPs\nwere identified by LC-MS-MS since their molecular weights did not match those\nof the identified impurities. Five DPs were found using LC-PDA, and total nine\nDPs were found by LC-MS-MS analysis, including DP1 and DP3. Different masses\nwere observed in LC-MS-MS for individual stress degradation condition which\ngives same retention time in LC-PDA when the mixtures of DPs are injected (peak\npurity studies reveled that some peaks were not pure in LC-PDA when mixture of\ndegradants was injected). Oxidative degradation showed different masses of\npeaks that had same retention time in LC-PDA as photolytic; neutral; thermal humidity;\nacid and base degradation. Hence for degradants observed in LC-MS-MS different\nnomenclature have been used for DPs. For oxidative degradation these were named\nDP6; DP7; DP8 and DP9. Also the peak of DP2 that was observed as single pure\npeak in LC-PDA (in case of acid, base, photolytic degradation) and was observed\nas bifurcated peak in LC-MS-MS with same MW hence two different nomenclature DP2\nand DP2 (e) was used during explanation. <\/p>\n\n\n\n

For\nPDM and DPs, the fragmentation paths and structures were predicted using the\nm\/z values. Figure 2a and 2b show the\nESI-MS-MS spectra of the DPs. Figure 3 depict the possible fragmentation\npathways of DPs.<\/p>\n\n\n\n

DP\n1 (m\/z 128): <\/em>There was an abundance of [M-H]–<\/sup>\nion at m\/z 128(127.92)  in the\nnegative ion ESI-MS of DP1; corresponding to MW of INH-1 Pyroglutamic acid. The\nMS-MS spectra of DP1 could not be generated due to low sensitivity and absence\nof ionization. <\/p>\n\n\n\n

DP2\nand DP2 (e) (m\/z 263): <\/em>The ESI-MS-MS\nspectrum of the ion at m\/z 263 gives two different fragmentation patterns that\nwas detected as a shouldered peak in LC-MS-MS while it was recorded as a single\npeak in LC-PDA. A possible explanation is the existence of an isomeric peak.\nDue to the loss of H2O, both had an abundance of fragment ions at m\/z of 245.\nThere was also an abundance of the fragmentation product (DP-2(e)) at m\/z 227)\nas well as DP-2(m\/z 148).<\/p>\n\n\n\n

DP3\n(m\/z 134): <\/em>The abundant [M+H]+<\/sup> ion was\nobserved at m\/z of 134 in the positive ion ESI-MS, that did not show protonated\nions. The MW of parent ion confirmed the anticipated structure for DP3 (table2)\nthat is thiazolidine carboxylic acid; the process related impurity (INH-2) of\nPDM.<\/p>\n\n\n\n

DP\n4 (m\/z 245): <\/em>The [M+H]+<\/sup> ion at m\/z 245\nwas prevalent in the positive ion ESI-MS-MS of DP4. The loss of H2O in the\n[M+H]+<\/sup> ion’s ESI-MS-MS spectra revealed  abundant fragment ion at m\/z 227. The spectra\nalso contains an abundant fragment ion of INH-2 (Thiazolidine\ncarboxylic acid), corresponding to m\/z 134.<\/p>\n\n\n\n

DP\n5 (m\/z 119): The <\/em>abundance of [M-H]–<\/sup>\nion at m\/z 119, was observed in the negative ion ESI-MS spectrum of DP5, which\ndid not show protonation.<\/p>\n\n\n\n

DP\n6 (m\/z 261): <\/em>The [M+H]+<\/sup> ion at m\/z 261\nwas prevalent in the positive ion ESI-MS-MS spectra of DP6. As shown by the\nESI-MS-MS spectrum, the most abundant [M+H]+<\/sup> ion fragment ion was\nfound to be the PDM or DP4 fragment ion, which fragmented to generate fragment\nion at m\/z 132.<\/p>\n\n\n\n

DP\n7 (m\/z 267): <\/em>There was an abundance of [M+H]+ in the\nDP-9 positive ion ESI-MS-MS spectra, which exhibited a m\/z 267. An abundant\nfragment ion at m\/z 261 found in the spectra of DP7, probably belong\nto DP6. These fragment ions further fragment to generate the most abundant\nfragment ions at m\/z 245 which correspond to either PDM or DP-4.Fragment ion m\/z\n245 further fragment to give m\/z of 148 that fragment to give m\/z of 132.<\/p>\n\n\n\n

DP\n8 (m\/z 521): <\/em>The positive ion DP8’s ESI-MS-MS\nspectra revealed an abundance of the [M+H]+<\/sup> ion at m\/z 521, which\nmay be the result of dimerization (m\/z 261) of DP8 causing the [M+H]+<\/sup>\nion abundance. The [M+H]+<\/sup> ion of DP10 has fragment ions of m\/z 485\nand 243 in the ESI-MS\/MS spectra.<\/p>\n\n\n\n

DP\n9: <\/em>The ACN adduct of DP8 was observed at\nRt 2.9 in oxidative degradation that gave [M+H]–<\/sup> ion in negative ion\nESI-MS spectrum at m\/z 306.<\/p>\n\n\n\n

Finally, structures and degradation\nschemes for all DPs were presented based on the aforementioned findings and\ninterpretations. TABLE 2 shows the chemical structures of PDM and all of its\nrelated compounds (both known and unknown), as well as their origins,\ndegradation pathways, Rt, and observed m\/z values of major fragments.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: a) ESI-MS-MS spectra of PDM and DPs<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 2b) ESI-MS-MS spectra of PDM and DPs<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 3: Anticipated fragmentation pathway of PDM and DPs<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Table 2: PDM and related molecules chemical structures, origin, degradation process, Rt, and measured m\/z values for main fragments.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

LC-PDA detection and\nLC-MS-MS investigation were employed during the profiling to track the\nimpurities and determine the degradability of Pidotimod. LC-PDA detected\nfive impurities in total, including degradation related and intrinsic or\ninherent impurities, while LC-MS-MS detected nine. Two degradation\nproducts produced during stress degradation were identified as inherent\/process\nrelated impurities INH-1 and INH-2. Out of nine impurities identified, six\nimpurities are not reported in literature. On the basis of LC-MS-MS analyses,\nstructures, fragmentation pathways, and a degradation strategy for degradation\nproducts were presented. This provides all of the data about Pidotimod’s\ndegradation chemistry.<\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

None to declare<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors have no financial or other conflicts of interest. <\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

There is no funding sources.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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