Acta Chromatographica DOI: 10.1556/1326.2017.29.1.06 Original Research Paper HPLC Determination of K027 in the Body of Pregnant Mice S.M NURULAIN1,2, S OJHA1, S DHANASEKARAN1, K KUČA3, N NALIN1, C SHARMA4, A ADEM1, AND H KALÁSZ1,5,* Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates Present affiliation: COMSATS Institute of Information Technology, Park Road, Islamabad, Pakistan 3Biomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic 4Department of Internal Medicine, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates 5Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Budapest, Hungary *E-mail: drkalasz@gmail.com Summary Distribution of K027, a hydrophilic, positively charged compound is monitored in the body of pregnant mice using high-performance liquid chromatography (HPLC) Intraperitoneal injection was done on the 18th day of pregnancy; the plasma and brains of the mother mice, placentae and the fetuses’ brains were dissected following 5, 15, 30, 60, and 120 of treatment Significant incorporation of K027 was found in the placentae and in fetuses’ brains relative to its levels in the mothers’ plasma and brains This incorporation warns of a possible adjustment of dose of pyridinium aldoxime antidotes in case of pregnancy Further studies with different gestational periods and animal models are warranted Key Words: K027, drug distribution, pregnant mice, pups, blood–placenta distribution, HPLC Introduction Penetration of xenobiotics into the central nervous system has been dealt with for several decades [1] Lipophilicity is an essential condition to indicate the extent of ability to penetrate The more lipophilic is the compound, its penetration is more expressed The discovery of carriers essentially changed both the situation and the explanations of blood–brain barrier transfer Even functions and penetration possibilities of other barriers (such as the placenta and the testis) are less frequently treated in publications Organophosphates and organophosphonates, such as pesticides, insecticides, and nerve agents, are used worldwide Their frequent use suggested ISSN 2083-5736©2016 The Author(s) S.M Nurulain et al the necessity of effective antidotes following poisoning [2] Application of an anticonvulsant, muscarinic receptor antagonist, electrolyte, and oxygen seems to be the general way of treatment A monopyridinium aldoxime, pralidoxime, has been used for a long time [3–6] Application of bispyridinium aldoximes (obidoxime and methoxime) has also been approved Kuca et al [7–13] synthesized a wide-range of bispyridinium monoaldoximes to improve the antidotal efficiency of the earlier used pyridinium aldoximes One of these new compounds was K027 (Fig 1), a bispyridinium monoaldoxime [7] Considering lipophilicity of these compounds, K027, K048, and K203 show the upmost negative log P as well as the largest total polar surface area (TPSA) values [14–16] O HO N NH N+ Br- N+ Br- Fig The chemical structure of K027 Tekes et al [17, 18] determined the level of K027 in serum, the brain, cerebrospinal fluid, and urine following i.m treatment of male Wistar rats The samples were treated using perchloric acid (PCA), and the HPLC separation was monitored using amperometric detection The calibration curve was linear between 10 and 250 ng mL−1, with a LOD of 10 ng mL−1 This LOD of amperometric detection resulted in an about 1000-fold increase in sensitivity compared to that of ultraviolet (UV) detection of either K027 or K048 and K203 [17, 19–23] Szegi et al [23] critically evaluated the HPLC analysis of several pyridinium aldoximes, both the classical (pralidoxime and obidoxime) and the newly synthesized ones (K027, K048, K074, K075, K203, and K1000) They also showed the effect of an ion-pairing agent OSA to be used Without OSA, each of the above-mentioned pyridinium aldoximes was eluted in the stationary phase (Zorbax Rx-C18) without any retention, while the to 14 mM OSA affected the peaks of the pyridinium aldoximes to have the appropriate retention Concerning K203 in the brain, an adequate separation can be reached using a relatively high concentration of OSA (2.5 g L−1) in the mobile phase, as the peak of K203 was interfering with the peaks of the brain matrix using g L−1 OSA in the mobile phase [23] HPLC Determination of K027 The objective of the paper was to find whether placenta acts as a barrier to penetrate the newer bispyridinium aldoxime, K027, and quantify the experimental aldoximes in developing fetus’ brain This study is the first one to report on this subject It is noteworthy that aldoximes are AChE reactivator AChE is the primary enzyme that serves to hydrolyze choline esters; especially, acetyl choline and AChE have been reported to play significant role in developing embryo and fetus brain, apart from the detoxifying property for OP poisoning Experimental Materials and Methods Chemicals Solvents and other chemicals were purchased from chemical sources in adequate (e.g., HPLC) qualities K027 (Fig 1) was the kind gift of Dr Kamil Kuca Animals and treatment The strain and origin of mice and the ethical permission (United Arab Emirates University Ethics Committee (IAEC/13/03) were detailed in our present publication [24] Pregnant mice on the GD18 were i.p (intraperitoneally) injected with 170 mg kg−1 of body weight of K027 solution in physiological saline Sample preparation Brain and placenta tissues of mice were dissected One hundred milligrams aliquots of the samples were mixed with 0.45 mL of phosphate buffer at pH 2.6, homogenized in an Ultra Turrax tissue homogenizer (IKA®-Werke GmbH & Co KG, Germany) for and at 10,000 rpm thermostated with ice bath Following homogenization, 0.3 mL of phosphate buffer and 0.2 mL of tricholoroacetic acid of 10% were also added and thoroughly mixed, and the precipitate was allowed to sediment by centrifugation at °C (for at 5000 rpm) The supernatants were used for determination Plasma samples were also collected They were mixed to 10% trichloroacetic acid (5:2 v/v ratio), and the precipitate was centrifuged for 10 at 10,000 rpm Supernatants were injected into the HPLC system without any further treatment — neither dilution nor concentration was done S.M Nurulain et al HPLC E2495 Alliance Waters setup was used with a built-in autosampler Detection was done at 275 nm HPLC separations were carried out using a Supelcosil octyl silica stationary phase (250 mm × 4.6 mm, i.d × μm) purchased from Supelco, Bellefonte, PA, USA; the mobile phase was an aqueous phosphate buffer (at pH 2.6 after adjustment with phosphoric acid) also containing an 8% methanol organic modifier, a 1-M 1-octane sulfonic acid ionpairing agent Mobile phase flow rate was mL min−1, and the sample size was 10 μL Validation of HPLC determination of K027 LOD and LOQ were 0.851 and 2.578 μg mL−1 Intra-day precision (Table I/A) and inter-day precision (Table I/B) were found adequate Table I/A Intra-day (six injections) precision of HPLC determinations Concentration added (μg mL−1) Average area ± standard deviation [AU × min] Concentration found (μg mL−1 ± standard deviation) RSD (%) Recovery (% ± SD) 0.5 16,392 ± 2875 0.40 ± 0.023 5.862 97.5 ± 6.5 107,191 ± 2014 4.53 ± 0.060 1.329 98.5 ±3.7 10 226,002 ± 2062 10.04 ± 0.097 0.970 98.8 ± 2.8 Table I/B Inter-day (three injections at the 1st, 2nd, and 3rd day) precision of HPLC determinations Concentration added (μg mL−1) Average area ± standard deviation [AU × min] Concentration found (μg mL−1 ± standard deviation) RSD (%) Recovery (% ± SD) 0.5 18,511 ± 2078 0.48 ± 0.01 2.022 98.7 ± 4.8 106,939 ± 4992 4.81 ± 0.09 1.784 96.9 ± 5.6 10 252,575 ± 5244 9.70 ± 0.37 3.778 99.5 ± 1.2 HPLC Determination of K027 Results A series of chromatograms of K027 in mother’s brains and plasma, in pup’s brain, and in placenta are given in Fig Its HPLC analysis shows linearity in the range of 0.5 to 5000 μg mL−1 range The calibration curve is in Fig 3, having slope, Y intercept, R2, and % bias of 20,954, 831,928, 0.999, and 0.8, respectively Inter-day and intra-day reproducibility were satisfactory Robustness study was also done (not shown here) System suitability study was done using (six) parallel injections of 10 μg mL−1 standard samples, which resulted in an average peak area of 233,519 AU × with 1624 AU × SD and 0.70 %RSD Peak purity was verified by setting and comparing the ultraviolet spectrum at the maximum and at the standard deviation values of the leading and tailing parts of the chromatographic peaks (not shown here) Fig A series of chromatograms of K027 HPLC chromatograms of samples were recorded after 15 following i.p injection of K027 Stationary phase was Supelcosil octyl silica (250 mm × 4.6 mm, with particle size: μm); the mobile phase was an aqueous phosphate buffer (at pH 2.6 after adjustment with phosphoric acid) also containing an 8% methanol organic modifier, a 1-M 1-octane sulfonic acid ion-pairing agent Mobile phase flow rate was mL min−1 The sample size was 10 μL Column temperature was kept at 40 °C S.M Nurulain et al μg/mL Fig Calibration curve of K027 for the HPLC separation The peak linearity of K027 was determined by injecting at least two times various concentrations ranging from 0.05 μg mL−1 to 5000 μg mL−1, and the average peak areas (AU × time) were plotted y = 20,954,005x + 831,927,601; R2 = 0.999 Table II Mean level of K027 in the pregnant mice and pup Each value represents the average of two mice, independently treated and determined using HPLC, expressed in μg mL−1 (plasma) or μg g−1 (brain and placenta) Time means elapsed time-period after i.p treatment Time (min) Mother’s plasma (μg mL−1) SD Mother’s brain (μg g−1) SD Placenta (μg g−1) SD Pup’s brain (μg g−1) SD 313.16 86.26 3.23 0.64 62.82 11.80 18.95 0.89 15 642.67 100.12 4.99 0.80 73.50 9.88 20.32 0.91 30 209.67 65.75 3.66 0.59 113.20 14.89 26.22 2.12 60 113.56 16.22 3.45 0.58 122.98 17.77 31.82 1.79 120 26.42 4.55 3.60 0.93 40.34 4.85 20.52 0.71 HPLC Determination of K027 The overall results of the HPLC determinations are given in Table II The level of K027 showed a definite maximum at 15 in the mother’s serum of pregnant mice However, a delayed maximum was found at 60 both in placentae, and the pups’ brain It was followed by a delayed offset in both the brains and placentae of the mothers, as well as in the pups’ brains (Table II) Discussion Pregnancy condition exhibits altered physiological situations which is influenced by many indigenous biochemicals Moreover, chemicals exposed under this situation may not be limited to mother only rather developing fetuses may also have good/bad consequences Hence, it is of utmost importance to know the fate of ingested drugs under new physiological situations Organophosphorus compounds are lipophilic in nature and crosses the biological barriers like placenta For instance, organophosphorous compounds, such as diazinon, parathion, and methyl parathion have been reported to penetrate through the placenta and inhibit the acetylcholinesterase enzyme both of the maternal and fetal brains [2, 25] Monitoring penetration through the placenta and the antidote level in the brain has thereby basic importance HPLC is an adequate method for the determination of K027 in various body compartments of rats [17] Female Wistar rats were both intramuscularly and intraperitoneally treated with 22.31 mg (50 μmol) K027, sacrificed following 5, 15, 30, 45, 60, 120, and 240 of treatment The plasma K027 level versus time graphs showed a definite single maximum at 15 min, followed by a constant decline Lorke et al found that about 0.6% of K027 enters the brain [26–28] Blood–brain penetration depends on the dose of K027, as Nurulain et al [29] found in a systemic experimental series The larger is the dose, the higher is the degree of penetration [29] Anyway, they found the maximum of K027 level in the plasma at 15 min, while the maximum level of K027 in the brains showed a definite delay About 10% of K027 plasma concentration was detected in the brains at the peak level (30 min) when either 30 or 60 μmol was i.m injected into rats [29] Atropine also penetrates through the placenta [30]; it is one of the antidotes against poisoning by organophosphates and organophosphonates S.M Nurulain et al Conclusions The study concludes that K027 crosses the placenta barrier efficiently and reaches to fetus brain in copious quantity which was more than the mother’s brain Blood–brain penetration and penetration properties through the placenta have essential importance The results of our experimental trial suggest that the dose of a pyridinium aldoximes antidote should be carefully selected in the case of pregnancy Acknowledgement Thanks are due to Mr M Shafiullah and Mr J Horváth for their advice Dr Kalász contribution was sponsored by OTKA 100155 grant of Hungarian National Scientific Granting Agency (Budapest, Hungary), and Dr Ojha’s contribution was supported in part by grants from United Arab Emirates University and United Arab Emirates National Research Foundation References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] P Aarseth and J.A Barstad, Arch Int Pharmacodyn Ther., 176, 434 (1986) A.W Abu-Quare and M.B Abou-Donia, J Appl Toxicol., 21, 307 (2001) G.A Petroianu, K Arafat, K Kuca, and J Kassa, J Appl Toxicol., 26, 64 (2006) G.A Petroianu, S.M Nurulain, N Nagelkerke, M.A Al-Sultan, K Kuca, and J Kassa, J Appl Toxicol., 26, 262 (2006) G.A Petroianu, M.Y Hasan, S.M Nurulain, N Nagelkerke, J Kassa, and K Kuca, Toxicol Mech Methods, 17, 401 (2007) S.M Nurulain, Trop J Pharm Res., 10, 341 (2011) K Kuca, J Bielavsky, J Cabal, and M Bielavska, Tetrahedron Lett., 44, 3123 (2003) K Kuca and J Cabal, Cent Eur J Public Health, 12 Suppl, S59 (2004) K Kuca and J Kassa, Vet Hum Toxicol., 46, 15 (2004) K Kuca and J Kassa, Hum Exp Toxicol., 23, 167 (2004) K Kuca, L Sevelová-Bartosová, and G Krejcova-Kunesova, Acta Medica (Hradec Kralove), 47, 107 (2004) K Kuca, J Cabal, and J Kassa, J Toxicol Environ Health A, 68, 677 (2005) K Kuca, K Musilek, D Jun, M Pohanka, K.K Ghosh, and M Hrabinova, J Enzyme Inhib Med Chem., 25, 509 (2010) J.Z Karasova, J Chladek, M Hroch, F Josef, D Hnidkova, and K Kuca, J Appl Toxicol., 33, 18–23 (2013) J.Z Karasova, F Zemek, K Musilek, and K Kuca K Neurotox Res., 23, 63 (2013) H Kalász, S.M Nurulain, G Veress, S Antus, F Darvas, E Adeghate, A Adem, F Hashemi, and K Tekes, J Appl Toxicol., 35, 116 (2015) HPLC Determination of K027 [17] K Tekes, M.Y Hasan, R Sheen, K Kuca, G Petroianu, K Ludányi, and H Kalász, J Chromatogr A., 1122, 84 (2006) [18] M Gyenge, H Kalász, G.A Petroianu, R Laufer, K Kuca, and K Tekes, J Chromatogr A., 1161, 146 (2007) [19] T Csermely, H Kalász, G Petroianu, K Kuca, F Darvas, K Ludanyi, A.A Mudhafar and K Tekes, K Curr Med Chem., 15, 2401 (2008) [20] H Kalász, M.Y Hasan, R Sheen, K Kuca, G Petroianu, K Ludányi, A Gergely, and K Tekes, Anal Bioanal Chem., 385, 1062 (2006) [21] H Kalász, J Fürész, and K Mini Rev Med Chem., 9, 596 (2009) [22] H Kalász, S.M Nurulain, G Veress, S Antus, F Darvas, E Adeghate, A Adem, F Hashemi, and K Tekes, J Appl Toxicol., 35, 116 (2015) [23] P Szegi, H Kalász, R Laufer, K Kuca, and K, Tekes, Anal Bioanal Chem., 397, 579 (2010) [24] S Ojha, S.M Nurulain, S Dhanasekaran, N Nalin, A Adem, C Sharma, and H Kalász, Blood-fetus penetration of pralidoxime, submitted for publication (2015) [25] S.M Nurulain, K Tekes, S.N.H Naqvi, C Sharma, and S Ojha, Arch Toxicol., 88, 575 (2014) [26] D.E Lorke, M.Y Hasan, S.M Nurulain, R Sheen, K Kuca, and G.A Petroianu, J Appl Toxicol., 27, 482 (2007) [27] D.E Lorke, H Kalász, G Petroianu, and K Tekes, Curr Med Chem., 15, 743 (2008) [28] D.E Lorke, S.M Nurulain, M.Y Hasan, K Kuca, K Musilek, and G.A Petroianu, J Appl Toxicol., 28, 920 (2008) [29] S.M Nurulain, H Kalász, P Szegi, K Kuca, A Adem, M.Y.B Hasan, F Hashemi, and K Tekes, Acta Chromatogr., 25, 703 (2013) [30] http://www.chemm.nlm.nih.gov/countermeasure_atropine-sulfate.htm Glossary AChE: acetylcholinesterase enzyme (EC:3.1.1.7) AU: absorbance unit at 275 nm GD18: gestation day of 18 HPLC: high-performance liquid chromatography i.m.: intramuscular (injection) i.p.: intraperitoneal (injection) LOD: limit of detection LOQ: limit of quantitation OP: organophosphorus OSA: octane sulfonic acid RBC: red blood cell SD: standard deviation TPSA: total polar surface area (Å2) UAE: United Arab Emirates UV: ultraviolet ... 0.71 HPLC Determination of K027 The overall results of the HPLC determinations are given in Table II The level of K027 showed a definite maximum at 15 in the mother’s serum of pregnant mice However,... g L−1) in the mobile phase, as the peak of K203 was interfering with the peaks of the brain matrix using g L−1 OSA in the mobile phase [23] HPLC Determination of K027 The objective of the paper... 1.2 HPLC Determination of K027 Results A series of chromatograms of K027 in mother’s brains and plasma, in pup’s brain, and in placenta are given in Fig Its HPLC analysis shows linearity in the