Applicability of LC-QToF and Microscopical Tools in Combating the Sophisticated, Economically Motivated Adulteration of Poppy Seeds

Morphine and codeine are the two principal opiates found in the opium poppy (Papaver somniferum L.) and are therapeutically used for pain management. Poppy seeds with low opiates are primarily used for culinary purposes due to their nutritional and sensory attributes. Intentional adulteration of poppy seeds is common, often combined with immature, less expensive, exhausted, or substituted with morphologically similar seeds, viz., amaranth, quinoa, and sesame. For a safer food supply chain, preventive measures must be implemented to mitigate contamination or adulteration. Moreover, the simultaneous analysis of P. somniferum and its adulterants is largely unknown. Pre- and post-processing further complicate the alkaloid content and may pose a significant health hazard. To address these issues, two independent methods were investigated with eight botanically verified and fifteen commercial samples. Microscopical features were established for the authenticity of raw poppy seeds. Morphine, codeine, and thebaine quantities ranged from 0.8–223, 0.2–386, and 0.1–176 mg/kg, respectively, using LC-QToF. In most cases, conventional opiates have a higher content than papaverine and noscapine. The analytical methodology provided a chemical profile of 47 compounds that can be effectively applied to distinguish poppy seeds from their adulterants and may serve as an effective tool to combat ongoing adulteration.


Introduction
Poppy seeds are a product of the opium poppy (Papaver somniferum L.), which belongs to the Papaveraceae family. P. somniferum has been cultivated for its seeds and their recreational, analgesic, and narcotic properties since prehistoric times [1,2]. These are tiny, edible, kidney-shaped seeds harvested as a source of alkaloid compounds for the pharmaceutical industry and as a source of poppy seeds for the food industry [3]. The sap, or latex, of the poppy plant, when dried to yield opium, contains multiple alkaloids, including opiates (OAs) such as morphine, thebaine, codeine, papaverine, and narcotine [4,5]. About 218 species belong to the genus Papaver [6,7], and 170 alkaloids have been reported from this genus [8]. Among these species, only Papaver somniferum L. and Papaver setigerum DC. (a synonym of Papaver somniferum subsp. setigerum (DC.) Arcang.) [9][10][11][12] produce higher quantities of narcotic substances as their secondary metabolites. Hence, the former species has been commonly cultivated. Although the seeds may contain low levels of opiates, contamination of residual poppy latex during harvesting and processing is known to contribute to higher levels of opiates [3][4][5]13]. three white poppy seed samples, ground seed samples, one ready-to-use poppy seed filling for the bakery, and two ready-to-eat products were analyzed for six alkaloids. All analyzed samples contained morphine between 0.2-241 mg/kg. Codeine (<0.1-348 mg/kg) and thebaine (<0.1-106 mg/kg) were identified in 78% of the samples. In four samples of black poppy seeds, Hayes et al. (1987) [25] found morphine and codeine in the range of 17-294 mg/kg and 3-14 mg/kg, respectively. The results obtained by Starnska et al. (2013) [39] indicated that the alkaloids morphine, codeine, narcotine, papaverine, and thebaine ranged from 3327-17175, 172-1767, 24-1675, 2-689, and 0-1701 µg/g, respectively, in the selected 15 poppy cultivars.
Sometimes, to increase profits, there is a possibility that the poppy seeds have been adulterated with locally available, less expensive seeds such as amaranth, cumin, chia, sesame, or other seeds that resemble poppy seeds in color and size, thereby increasing the difficulty of determining the authenticity of the poppy seeds, including artificially colored seeds [47,48]. In addition, DNA barcoding methods were successfully implemented to detect adulterants of the Papaver genus and helped differentiate P. somniferum from other varieties [49,50]. Poppy seed pods are harvested to extract opiate-containing latex before maturity, while culinary poppy seeds are harvested after the seed pods have matured and dried. There have been cases of poppy seed adulteration when immature by-product seeds from pharmaceutical processing with a higher content of opium alkaloids were mixed with food-grade mature poppy seeds. Often, the poppy seeds are heterogenous, having differences in size, color, and taste, and may be contaminated with poppy latex or other impurities [51].
Culinary poppy seeds are frequently mixed with low-quality seeds of cultivars with higher alkaloid content or with exhausted seeds following the extraction of opiates. The mixed product's decreased quality and increased OA content represent a potential health hazard for consumers [19,52,53]. Even if establishing the detailed external morphological features of botanically verified materials would serve for the proper identification of raw materials, the development of a sensitive analytical method would address the overall quality of the poppy seeds in commerce or in poppy-derived products. Moreover, assessing extraction efficiency with various solvents and removing opiates from post-processed poppy seeds would be beneficial to differentiate naturally latex-contaminated seeds from seeds artificially spiked with opiates. Nevertheless, an untargeted analysis and the development of comprehensive alkaloid profiles could help discriminate between species in seeds of the genus Papaver and for detecting any added adulterants or substituents.

Chemicals and Standards
Representative images of poppy seeds and the chemical structures of five OAs are shown in Figures 1 and 2. Morphine, codeine, thebaine, noscapine, papaverine, morphined 3, and codeine-d 3 were purchased from Sigma (St. Louis, MO, USA). The reference compounds were more than 99% pure, with identity and purity confirmed by chromatographic and spectral data (HRMS). HPLC-grade solvents such as acetonitrile, methanol, isopropanol, and formic acid were procured from Fisher Scientific, Waltham, MA, USA. Ultrapure water (18.2 MΩ.cm) was purified using the Milli-Q system (Millipore, Bedford, MA, USA).

Preparation of Standard Solutions
Stock solutions of standard compounds were prepared at a concentration of 1 µg/mL in methanol. Calibration curves ranged from 0.5-250 ng/mL at five different concentration levels. All standard solutions were stored in amber vials at 4 • C. Poppy seed samples (#2116-2021, 2123, and 2124) used in food were commercially procured from Amazon in November 2022. These samples were deposited at the NCNPR's botanical repository, the University of Mississippi, MS, USA.

Plant Sample Preparation
Dry-ground seed samples were weighed separately for about 1000 mg, 100 mg, and 10 mg each. 100 µL of the internal standards (2.5 µg/mL) were added to these samples and sonicated in a 2.5 mL volume of methanol containing 1% formic acid for about 30 min, followed by their being centrifuged for 15 min at 959× g. The supernatant solution was transferred to a 10 mL volumetric flask. The procedure was repeated three more times with fresh solvent, and their supernatants were combined in a volumetric flask and adjusted to achieve a final volume of 10 mL. Before injection, approximately 2 mL of the solution was passed through a 0.45 µm PTFE membrane filter. The first 1.0 mL was discarded, and the remaining volume of about 1 mL was collected in an LC sample vial.

Micro Morphology Analysis
The external morphology of seeds was observed under low magnification using a NIKON SMZ-U (Japan) stereomicroscope with photo capture by the camera (NIKON DS-Fi1) attached to the microscope and processed with the software NIS-Element BR. Specimens were fixed in formaldehyde alcohol acetic acid (FAA) for high-level magnification using scanning electron microscopy (SEM) analysis. These samples were passed through a 100% ethanol solution and dehydrated using a 1 h and 15 min programmed critical point dryer (Leica CPD300, Wetzlar, Germany) under CO 2 . Dried samples were placed on double-sided adhesive carbon tape and pasted on the aluminum stubs. For platinum coating, these stubs were placed on a Desk V HP sputter coater (Denton Vacuum, NJ, USA) supplied with argon gas. The prepared samples were used for SEM analysis using a JSM-7200FLV field-emission SEM (JEOL Ltd., Tokyo, Japan).

Instrumentation and Analytical Conditions
Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry (LC-QToF) The liquid chromatographic system was an Agilent Series 1290 system comprised of a binary pump, a vacuum solvent micro-degasser, a 100-well autosampler, and a thermostatically controlled column compartment. Separation was achieved on an Agilent Poroshell 120 EC-18 (2.1 × 150 mm, 2.7 µm) column at a 0.2 mL/min flow rate. The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) with the following gradient elution: 99% A/1% B, isocratic for 3 min, 45% B in the next 27 min, and 100% B in the next 10 min. A 5 min wash followed each run with 100% acetonitrile and an equilibration period of 5 min with 99% A/1% B. One microliter of the sample was injected at a column temperature of 40 • C. The mass spectrometric analysis was performed with a QToF-MS/MS (Model #G6545A, Agilent Technologies, Palo Alto, CA, USA) equipped with an ESI source under the following conditions: drying gas (N 2 ) flow, 13.0 L/min; drying gas temperature, 325 • C; nebulizer, 27 psig; sheath gas temperature, 325 • C; sheath gas flow, 11 L/min; capillary, 3500 V; skimmer, 65 V; Oct RF V, 750 V; and fragmentor voltage, 150 V. The sample collision energy was set at 45 eV. All operations, data acquisition, and data analysis were controlled by Agilent MassHunter Acquisition Software Ver. A.10.00, with further data processing by MassHunter Qualitative Analysis Software Ver. B.10.0. Each sample was analyzed in positive and negative modes in the range of m/z = 50-1700. Accurate mass measurements were obtained employing ion correction methodologies using mass references at m/z 121.0509 (protonated purine) and 922.0098 [protonated hexakis (1H, 1H, 3H-tetrafluoropropoxy) phosphazine or HP-921] in positive ion mode, and mass references at m/z 112.9856 (deprotonated trifluoroacetic acid-TFA) and 1033.9881 (TFA adducted HP-921) were used in negative ion mode. The compounds were confirmed in each spectrum.

Method Validation
The LC-QToF method was validated following International Conference on Harmonization (ICH) guidelines regarding precision, accuracy, carryover, stability, and linearity [54]. The limit of detection (LOD) and limit of quantification (LOQ) parameters were determined by injecting a series of diluted solutions with known concentrations. LOD and LOQ were defined as signal-to-noise ratios equal to three and ten, respectively. The accuracy of the assay method was verified in duplicate using two concentration levels of 10 and 100 ng/mL. Intra-and inter-day variation of the assay was determined on three consecutive days with three repetitions each [55]. To understand the possible role of grinding poppy seeds, 100 mg of intact seeds (#2120PR) and the corresponding ground mixture (#2120PR) were extracted separately with 1% formic acid in methanol (10 mL) in five replicates and analyzed for the content of the five alkaloids: morphine, codeine, thebaine, papaverine, and noscapine.

Washing Effect on Poppy Seeds
To understand the possible impact on OAs content of water washing intact poppy seeds, 500 milligrams of poppy seeds (#2546) were soaked in water (10 mL) and methanol with 1% formic acid (10 mL) separately at different temperatures (cold and hot, 70 • C) and at different time intervals (1, 10, 30, 60, and 120 min; 4, 8, 24, and 48 h; and 4, 6, and 8 days) with constant agitations. At each time increment, 100 µL of an aliquot was collected and analyzed for morphine, codeine, thebaine, papaverine, and noscapine. At the same time, 100 µL of water or methanol (1% formic acid) was added to maintain the constant final volume (10 mL). After 8 days of washing, all samples were dried, ground, and extracted with 10 mL water or methanol (1% formic acid) separately and analyzed. Effects of solvent, temperature, and extraction time factors on poppy seeds were also investigated.
In another experiment, ground poppy seeds (500 mg, #2546) were soaked in 10 mL each of water and methanol (1% formic acid) at different temperatures (cold and hot, 70 • C) and at different time intervals (1, 10, 30, 60, and 120 min; 4, 8, 24, 48 h; 4, 6, and 8 days) with constant agitations. At each time, 10 mL of solution was removed and analyzed for morphine, codeine, thebaine, papaverine, and noscapine. At the same time, 10 mL of water or acidified methanol was added. This process was continued for 8 days, and after 8 days of thorough washing, the samples were dried and ground. Samples were again extracted with water or methanol (1% formic acid) and analyzed separately. In this study, the OAs in each washing cycle were estimated.

Results and Discussion
3.1. Scanning Electron Microscopic Observation of Poppy and Its Adulteration/Substitution Seeds P. somniferum seeds are of a tiny reniform shape and are noticeably blue/black and white-colored. Seeds are, on average, 1.65 mm long and 1.15 mm wide. The different color seeds are morphologically similar, with white seeds being 0.10-0.25 mm smaller than black seeds. Blue/black/white seeds show a rough surface with a pitted honeycomb-like structure, as shown in Figure 3. The surface is rough in blue-black seeds (Figure 3a-f), whereas in white seeds, the texture is less waxy coated (Figure 3g Table 1. Microscopically, these adulterant seeds have different seed coat (testa) textures from genuine poppy seeds. Eschscholzia californica seeds have a rough and microsculpted surface, but they can be differentiated by the absence of the characteristic honeycomb texture observed in poppy seeds (Figure 3m    ; #25107 (f,f'); #16725 (g,g'); and #5558 (h,h') show the whole seed view and testa texture with a reticulate waxy plate in blue/black/brown seeds and mild in white seeds. The adulterant samples: N. sativa (i,i') has a reticulate pattern of ridges with an ocellate spinulose testa texture; C. quinoa (j,j') has a "dusty" surface, with no protuberances; S. indicum (k,k') has a smooth texture with some nerve lines; S. hispanica (l,l') has a smooth texture; E. californica (m,m') has a rough and micro sculpted texture; A. cruentus (n,n'), A. caudatus (o,o'), are with smooth texture; A. paniculatus (p,p'), has a reticulate somewhat hierarchically structure. Scale bars are marked respectively for each image.

Extraction from Poppy Seeds
Extraction efficiency for alkaloids was investigated using the following solvents and solvent mixtures covering a broad range of polarity index values: methanol, acidified methanol (0.1% and 1% formic acid), ethanol, water, and solvent mixtures such as acetonitrile-water (1:1, v/v), acetonitrile-water (1:1, v/v) with 0.1% formic acid, IPAmethanol (1:1, v/v), and IPA-ethanol (1:1, v/v) with 0.1% formic acid ( Figure 4). Poppy seeds (# 2117PR) were homogenized using a grinder. Approximately 100 mg of the seeds were weighed in duplicate, and the solvent was added. A mixture of deuterated internal standards (morphine-d3 and codeine-d3) was added to each solution; tubes were capped, placed into an ultrasonic bath for 30 min, and centrifuged at 4000 rpm for 15 min. Ultrasound-assisted extraction was carried out and optimized for the above solvents, time of extraction, and concentration of constituents in each organic solvent. Among the different solvents, the optimum extraction conditions were realized when 100 mg of poppy seeds were extracted with 10 mL of methanol containing 1% formic acid (see under the Section 2).

Optimization of Chromatographic Conditions
After several trials, optimized chromatographic conditions were achieved with acetonitrile and water with formic acid in different proportions for the mobile phase and column temperatures at ambient and 40 • C. Mass Hunter Workstation software, including Qualitative Analysis (version B.10.00), was used for processing both raw MS and MS/MS data. This includes molecular feature extraction, background subtraction, data filtering, and molecular formula estimation (using an exact mass of all isotopes, their relative abundances, and all detected adducts with their measured isotopes). A blank sample (methanol) was analyzed under identical instrument settings, and background molecular features (MFs) were removed. This method also involved using the [M + H] + ions in the positive ion mode found in the extracted ion chromatogram (EIC).

Validation Procedure
According to ICH guidelines (Table 2), the newly developed LC-QToF method for the five opiates (OAs) was validated for selectivity, sensitivity, the limit of detection (LOD), the limit of quantification (LOQ), stability, precision, accuracy, specificity, and linearity [54]. Regression equation The specificity of the method was determined by comparing chromatograms of the blank (methanol) with poppy seed samples and spiked blanks (OAs added to methanol and code #2124PR, 25107). A comparison of methanol blanks with samples and spiked blanks demonstrated the specificity and selectivity of the chosen methodology.
The LOD and LOQ were determined by injecting diluted solutions with known concentrations of each standard. LOD and LOQ were defined as signal-to-noise ratios equal to three and ten, respectively. A five-point calibration curve for the five opioid alkaloids showed a linear correlation between concentration and peak area. Calibration data indicated the linearity (r 2 > 0.99) of the detector response. The detection and quantification limits were 10-25 pg/mL and 25-100 pg/mL, respectively. All samples and standard solutions were analyzed in duplicate.
The accuracy of the assay method was evaluated by spiking two products (#2124PR and #25107) in duplicate using concentration levels of 10 and 100 ng/mL. The method's accuracy was determined for the related compounds by spiking the sample (#2124PR and #25107) with a known amount of the five opiates standard. These samples were spiked with known amounts of the standard compound mixture and were extracted four times under optimized conditions. These sample's percentage recovery (%RSD) ranged from 90-109% (1.7-4.4%).
The precision of a method is the degree of agreement among individual analytical results when the procedure is applied repeatedly to multiple samples of each product. The intra-and inter-day precisions were estimated by analyzing multiple replicates of two products (#2124PR and #25107). The intra-day precision of the assay was estimated by calculating the relative standard deviation (%RSD) for the analysis of samples in three replicates (n = 3) of each product, and the inter-day precision was determined by the analysis of three replicates of the same product on three consecutive days. The intra-day %RSD for the replicates was between 0.7 and 3.9%, and the %RSD for the day-to-day replicates was between 1.1 and 3.9%.
From the measured standard deviation (SD) and mean values, precision as relative standard deviation (%RSD) is calculated as %RSD = (SD/mean) × 100.
The sample solution (#2124PR and #25107) and standard solutions (10 and 50 ng/mL) were prepared by the proposed method and subjected to a stability study at room temperature for 72 h. The sample solution was analyzed at the initiation of the study period and at three additional time intervals before 72 h. No significant changes were observed.

Analysis of Poppy Seeds
LC-QToF-MS is a powerful technique offering high sensitivity and selectivity for qualitative and quantitative measurements of analytes of interest in a complex mixture. The high-resolution mass detection in the positive ion mode is useful for detecting alkaloids. In the positive ion mode, protonated species [M + H] + at m/z 286.1440, 300.1596, 312.1597, 340.1541, and 414.1545 for morphine, codeine, thebaine, papaverine, and noscapine, respectively, were observed. The combination of retention time and exact mass results are helpful for the general identification of the compounds. The calibration curves were prepared for the compounds listed in Table 2. The validation parameters-retention time, molecular formula, and characteristic fragment ions observed for all compounds-are summarized in Tables 3 and 4. Retention time, accurate mass, and MS/MS analysis are helpful in the identification of compounds. Table 3. Content of µg/g of different alkaloids in various ground poppy seeds using LC-QToF-MS.

No.
NCNPR # Content of Alkaloids (µg/g ± %RSD) Total Amt. of Five Alkaloids (µg/g ± %RSD) Morphine Codeine Thebaine Papaverine Noscapine * Mean values (n = 3) ± %RSD; DUL = detected under limits of quantification.   From each batch of poppy seeds, two different portions (each weighing ∼100 mg) were ground, extracted, and analyzed. Stranska et al. (2013) reported the alkaloids content from 15 different cultivar samples as 3327-17175 µg/g (morphine), 172-1767 µg/g (codeine), 0-1701 µg/g (thebaine), 2-689 µg/g (papaverine), and 24-1675 µg/g (noscapine) [39]. This is while Lopez et al. (2018) reported that the alkaloids' content varies from 0.2-241 mg/kg (morphine), <0.1-3.48 mg/kg (codeine), and <0.1-106 mg/kg (thebaine) in 41 different commercially purchased samples from the Netherlands, Germany, and Italy [19]. In our study, 23 poppy seed samples (15 black/blue/brown mix and 8 white/brown mix) were analyzed for the content of five OAs. 17 of the 22 samples showed higher morphine content than any of the other four OAs analyzed. In these 17 samples, the morphine content ranged from 0.8 to 223 µg/g. Two samples (#24792 and #2120PR) showed high codeine content, while codeine content ranged from 0.2-386 µg/g across all 22 samples. One sample (#2117PR) showed a high thebaine content, whereas the thebaine content ranged from 0.1 to 176 µg/g across all 22 samples. Higher concentrations of morphine, codeine, and thebaine were detected in black and black-blue mix samples, while these OAs were lower in white and white-brown mix samples. The other two compounds, papaverine, and noscapine, ranged from DUL-28 µg/g to DUL-73 µg/g, respectively. Sample #6485, labeled "Papaver species," contained all five OAs (Table 3) and showed a profile similar to that of P. somniferum. The mean levels of OAs are shown in Table 3, Figures 5 and 6. Differences in the cultivar varieties, harvesting conditions, and post-processing procedures (viz., washing) affect the alkaloids content from the previously published reports. Furthermore, compared to previous reports, the developed method can perform quantitative analysis of opiates as well as chemical profiling to differentiate authentic poppy seeds from adulterants. Nevertheless, high OAs variation in these samples suggests that these poppy seeds may not meet the limits set by European authorities.

Alkaloid Content in Ground vs. Intact Poppy Seeds
This experiment was aimed at evaluating alkaloid degradation during the processing of food. Unground and ground seeds were extracted separately with methanol (1% formic acid) in five replicates and analyzed for alkaloid content. The influence of grinding on the OA content was studied. Insignificantly lower concentrations of OAs were found in the ground seeds compared to the unground, intact seeds. Around 10-15% depletion of three alkaloids was observed, accounting for the formation of pseudomorphine (C 34  . The data also suggested that temperature and light have minor effects on the content of the five studied opiates. Besides the poppy variety, the harvest method has the strongest influence on the morphine content and leads to the greatest variability in the alkaloid concentration. High OAs concentrations can be attributed to insufficient harvesting practices leading to seed contamination with latex, inadequate cleaning and handling, or intentional spiking with pure standard morphine. The non-uniformity of this contamination leads to inhomogeneities observed within the sample replicates. Similar observations were seen by Sproll et al. (2006 and2007) [4,19,38,56,58]. When the levels of OAs in unground poppy seeds from five replicates within each batch (#2120) were compared, it was found that there was much variation with the levels of morphine, codeine, thebaine, and noscapine ranging from 0.6-7.6 µg/g, 218-424 µg/g, 12.4-68.2 µg/g, and 0.5-1.4 µg/g, respectively, suggesting possible anomalies in post-harvesting. While in the ground seed samples, there was little variation between the five replicates (#2120), indicating the homogeneity of ground samples. These wide variations observed within the same seed sample suggest the presence of OAs may be due to inadequate cleaning of the seeds, with most of the opium alkaloids coming from debris or residual latex on the seed surface.   Very low levels of OAs were detected in studied poppy seed samples using the current analytical methodology. This study confirms that poppy seed morphine or other OAs originate predominantly from external contamination. This was further confirmed by several seed-washing experiments that significantly reduced the OA content. Bjerver et al. (1982) [34] showed that 40% of the total morphine could be removed by a single washing with slightly acidified water. Soaking poppy seeds in water for five minutes removed about 46% of their free morphine and 48% of their codeine [59]. Moreover, the results presented in Figure 6 shows that about 40% of the total opioid content in the seed samples could be isolated from the seeds by a single, simple washing procedure. Alkaloid content in poppy seeds found in this study agrees with prior reports [34,56,59].

Identification and Characterization of Chemical Constituents
In this study, positive ESI tandem mass spectra of the [M + H] + ions of alkaloids were obtained using LC-QToF. High-resolution mass resolving power and accurate mass measurement for MS and MS-MS experiments enable rapid dereplication of various alkaloids within the genus Papaver. It generates characteristic fragment ions, which help confirm the known compound in a complex mixture. Possible fragment ions are shown in Table 4. Accurate mass measurements were performed to obtain each analyte's elemental composition. The diverse class of alkaloids, the major components of poppy seeds, have been categorized into seven groups based on their structural motifs and MS/MS fragmentation patterns, including morphinane, benzylisoquinoline, rhoeadine, tetrahydroisoquinoline, promorphinane, protopine, and benzophenanthridine types. The mass spectral fragmentation patterns of five reference compounds were studied, and based on these patterns, other alkaloids were investigated. Figure 8 shows the extracted ion chromatograms (EIC) of reference standards and poppy seeds (#24792) that have five opioids (OAs).  (Table 4) Full-scan mass spectra in the positive ion mode of the LC peak at 3.3 min provided a quasimolecular ion at m/z 154.0863, suggesting a molecular formula with [C 8 H 11 NO 2 ] + (calculated to be 154.0863). The MS 2 spectrum of the ion at m/z 154.0863 generated a series of ions at m/z 137.0598, 119.0488, 109.0647, and 91.0543, which agree with the fragmentation pattern of dopamine. The formation of product ions m/z 137 and 119 is attributable to the neutral loss of the amine group [M + H-NH 3 ] + and water molecule from the terminus of the dopamine molecule (Table 4) [60].
The major fragmentation pathway of protonated phenylalanine (m/z 166.0861) starts with the loss of water and CO to form a fragment ion at m/z 120.0801. Further loss of ammonia resulted in a fragment ion with m/z 103.0545. Leucine and isoleucine (m/z 132.1015) produced two fragments by sequential losses of H 2 O and CO (m/z 86.0966) and NH 3 (m/z 69.0696) [60].  (Table 4) Morphinane: The fragments are formed by cleavage of the piperidine ring and loss of an amine (CH 2 CHNHCH 3 , [M-57] + ). Such a fragment is found for all morphine-like compounds and is crucial in further fragmentation pathways [62].

Fragmentation Patterns of Alkaloids (Compounds 7-47)
Morphine and codeine exhibit similar fragmentation patterns, and the major product ions obtained were m/z 201.0910 and m/z 215.1067, respectively, which were derived from the precursor ion [M + H] + by cleavage of the piperidine ring and consecutive losses of ethylene methylamine [CH 2 CHNHCH 3 ] + and CO [62]. Consecutive losses of two water molecules in the case of morphine or methanol and water for codeine result in a cyclopentadiene naphthalene product ion with m/z 165.0704 and the elemental composition [C 13  is also detected, which can be explained by the loss of aziridine (m/z 43.0422, (CH 2 ) 2 NH) and water [64].
Benzyltetrahydroisoquinoline and aporphine alkaloids were distinguished by characteristic losses of the NHR 1 R 2 (R 1 and R 2 represent the substituent groups of the nitrogen atom) radical and the fragment ions below m/z 200. The MS/MS investigation of benzyltetrahydroisoquinoline and aporphine alkaloids results in fragment ions [M-45] + , [M + H-31] +, and [M + H-17] + always being observed initially depending on the number of N-methyl groups. These fragments are attributed to a loss of the NH(CH 3 ) 2 (R 1 = R 2 = CH 3 ), NH 2 CH 3 (R 1 = H, R 2 = CH 3 ), and NH 3 (R 1 = R 2 = H) radicals, respectively. These characteristic ions play a diagnostic role in the discrimination of other alkaloid types. However, the mass spectrum fragmentation behaviors of magnoflorine and corydaline were different from those of the benzyltetrahydroisoquinoline alkaloid because of the conjugate structure. No characteristic ions exist below m/z 200, and the product ions are formed mainly by the loss of some substituent groups [65].
Protopine alkaloids ( Sanguinarine (benzophenanthridine-type) was also characterized by the fragmentation of their substituents, with the most abundant ions being formed by the loss of CH 3 (m/z 317.0685), CH 2 O (m/z 302.0811), and CO (m/z 276). Benzophenanthridine alkaloids contain a large π conjugate system, making the parent nucleus difficult to fragment. The benzophenanthridine alkaloid sanguinarine, containing a methylenedioxy group, would lose carbon monoxide to form a stable ternary oxygen ring [67].
The can be rationalized by the losses of dimethylamine and one or two water molecules, respectively [19].
Benzylisoquinolines: These correspond to ion types a, [M + H-NH 3 ] + , and ion types b, c, the protonated parental molecule [M + H] + , and ion type d, respectively. The resulting ion at m/z 123.04390 constitutes the signature fragment obtained for 1-benzylisoquinolines containing hydroxyl groups at C3 and C4 . Increases in 14 or 28Da (m/z 137 or m/z 151) for the equivalent ions indicate single-or double-methylation at C3 and C4 [70].
Papaverine is a non-narcotic alkaloid found to be endemic and selectively localized in the latex of the opium poppy. Papaverine readily produced an m/z 340.1543 [M + H] + protonated pseudomolecular ion. The fragmentation of the precursor ion can be explained by the loss of a methyl group, resulting in a product ion at m/z 324.1236, and the loss of the dimethoxy benzyl moiety to yield the prominent isoquinoline moiety at m/z 202.0868 via the C-C bond between the dimethoxy benzyl and isoquinoline ring systems. The subsequent loss of a methoxy group results in one or more fragments with m/z 171.0682 [64]. The product ion at m/z 187.0628 was an isoquinoline ring, and the product ion at m/z 156.0443 was produced by demethylation of the fragment ion at m/z 171. The product ions and the corresponding neutral fragment loss noticed in our study agree with the work demonstrated by Wickens et al. (2006) [64] and Peng et al., 2007 [71] for the characteristic structural information of papaverine. 330.1700). The fragmentations indicated the initial loss of methylamine to form an ion of m/z 299.1281, followed by fragmentation of the benzyl moiety (m/z 137.0600). The stable naphthalene ion (m/z 175.0750) was formed from the rearrangement of the ion m/z 299. The benzylisoquinoline ion (m/z 192.1022) was formed directly from the quasimolecular ion [M + H] + and was the diagnostic ion for tetrahydroisoquinoline type of alkaloids [65,72].
Benzo[c]phenanthridines: Sanguinarine ([M] + ; m/z 332.0917) is benzo[c]phenanthridines, most of whose fragmentation ions form by cleavage of the substituted groups rather than ring fusion. As a derivative of protopine, sanguinarine contains two methylenedioxy rings. The base peak at m/z 304.0969 results from the rearrangement of one methylenedioxy ring and the consequent loss of CO. Consecutive losses of CH 2 O and CO generate other fragments at m/z 274.0865 and 246.0915, respectively [68].

Poppy Seed Adulteration or Substitution
Poppy seeds are relatively expensive and are sometimes mixed with seeds that closely resemble poppy seeds, such as seeds of Amaranthus species [48,73]. The adulterants are often similar in look, color, and texture to authentic poppy seeds. Even though some adulterant seeds may not present a health hazard, they are less expensive, and their use results in economic losses for food poppy seed producers and places fraudulent products in the marketplace.
Adulterated seeds were observed under 10× to 50× magnifications, and the salient features were noted to differentiate the adulterants from the genuine poppy seeds ( Figure 9 and Table 1). Poppy seeds from different cultivars vary in size, color, and appearance, with the black/blue/brown seeds having a rough waxy coat while the white seeds have a smooth non-waxy coat. Many adulterants, such as Amaranthus seeds, are used commercially to increase the weight of poppy seeds, leading to lower-grade materials in commerce. In this study, an LC-ESI-MS/MS technique provided applicable information to characterize 47 compounds from P. somniferum and major components from adulterants using authenticated plant materials. With this methodology, ground plant material could be analyzed to confirm or deny the presence of P. somniferum, which should aid in detecting adulteration or preventing the use of potentially mislabeled or misidentified "P. somniferum" material. Poppy seeds containing varying amounts of adulterants were analyzed. Adulteration as low as 10% could be easily detected based on the constituents shown in Table 5. Other possible adulterants of poppy seeds (Table 5) are:    Salvia hispanica, commonly known as chia, is a species of flowering plant of the mint family, Lamiaceae, native to Central and Southern Mexico and Guatemala [74]. Ground or whole chia seeds are consumed in Paraguay, Bolivia, Argentina, Mexico, and Guatemala for nutritious drinks and as a food source. S. hispanica is a source of omega-3 fatty acidslinolenic acid, linoleic acid, oleic acid, pantothenic acid, myricetin, the main flavanol, quercetin, and kaempferol are found in chia seeds.
Amaranthus caudatus, A. paniculatus, and A. cruentus: Amaranthus of the family Amaranthaceae, which includes quinoa and amaranth species, is a valuable food source of nutrients with high-quality proteins, vitamins, minerals, and bioactive compounds such as phenolics [81,82]. Approximately 35 to 55 Amaranthus species are native to the Americas, and at least 15 species are native to Africa, Europe, and Asia. Amaranthus is an important nutritional crop. It is a rare plant, with leaves consumed as a vegetable while seeds are eaten as a cereal [83,84]. A. caudatus, A. hypochondriacus, and A. cruentus, used for grain purposes, have tremendous potential to increase food production. Amaranth is commonly used in the bakery for cookies, biscuits, candies, pancakes, pasta, noodles, etc. [85]. In addition to its nutritional properties, amaranth offers an attractive source of lysine and other bioactive compounds such as phenolics, squalene, folate, phytates, and tocopherols [86]. Studies have also revealed the presence of olean triterpene saponins in the seeds.
Sesame (Sesamum indicum) has long been used extensively as a traditional food in the Middle East and South Asia. Sesame seed and sesame oil are widely used in cooking and as ingredients in sweet and confectionery foods. The potent antioxidant properties of seed extracts from S. indicum and sesame oil are attributed mainly to the presence of the lignans, including sesamin, sesamolin, sesamolinol, sesaminol, and lignan glycosides [87].
Chenopodium quinoa: Due to their high protein content, quinoa seeds have high nutritional value and are known to possess several flavonoids, including kaempferol glycosides, quercetin glycosides, and saponins (Table 5) [88].
Eschscholzia californica, the California golden poppy, California sunlight, or cup of gold, is a flowering plant in the family Papaveraceae, native to the United States and Mexico. The Californian poppy has a sap that contains a different class of alkaloids from that of the addictive "opium poppy" and does not have a narcotic effect on the human body. Similar to all poppy alkaloids, these have a sedative and relaxant effect on the body and mind, but they are perceived to be gentle and mild. Analysis showed the presence of morphinan alkaloids in opium poppy (P. somniferum) and benzophenanthridine alkaloids in E. californica. Several chemical compounds have been identified in E. californica, including californidine, cryptopine, eschscholtzine (californidine), sanguinarine, chelirubine, and other similar (Papaveraceae) alkaloids [89].

Conclusions
Two independent methodologies (microscopy and LC-QToF) were investigated with twenty-three poppy seed samples and eight adulterants. Detailed micromorphology observations with salient features highlighted may be used to differentiate poppy seeds from adulterant seeds. The developed LC-QToF method was identified as a sensitive and selective analytical method to determine five opiates in various colored and noncolored poppy seeds. The method was validated in terms of linearity, accuracy, selectivity, LOD, LOQ, and precision. A variation in total alkaloid content was noted among different color poppy seeds, specifically morphine, codeine, and thebaine, ranging from 0.8-223, 0.2-386, and 0.1-176 mg/kg, respectively. In most cases, the phenanthrene alkaloids morphine (dominant), codeine, and thebaine are in higher concentrations than the benzylisoquinoline alkaloids papaverine (<LOD-28 mg/kg) and noscapine (<LOD-73 mg/kg). The optimal treatment for reducing morphine and other OAs in poppy seed consists of washing, heating, and grinding. The application of LC-QToF provided useful information to characterize forty-seven compounds in poppy seed samples. Cleavage of the piperidine ring with consecutive losses of an amine and CO were some of the characteristic fragment ions for morphine, thebaine, and codeine, although this fragmentation process is not applicable for noscapine and papaverine. The developed simple and accurate method will be useful for determining and confirming unknown molecules, contaminants, or adulterants via a targeted or non-targeted approach. It is essential for identifying P. somniferum. There were no adulterants found in any of the poppy seed samples studied, which included seeds of amaranthus, golden poppy, black cumin, chia, sesame, and quinoa. However, the developed methods should serve as proactive tools to combat potential adulteration with seeds that closely mimic poppy seeds.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.