Abstract: Spagyric plant medicinerepresents a novel whole-plant medicine preparative methodology originatingfrom the Paracelsian alchemical tradition. Modern spagyric practitioners employthis methodology by incorporating recrystallized mineral salts derived from thewater-soluble ash component of a calcined plant material into the correspondingplant tincture. The mineral salt addition introduces essential nutritionalconstituents to the plant tincture which can be a source of therapeutic valuein spagyric plant-based medicines. The spagyric tincturing method often leadsto changes in color, consistency, and pH of the tincture’ constituents. It issuspected that alkali salification to raw typically acidic phytocompounds,especially in presence of alcohol, could lead to any number of intra-tincturechemical reactions. This addition of salt content and potential intra-tincturereactions can alter the chemical fingerprint leading to xenobioticphytochemistry and thus alternative pharmacological actions. To assure theidentity, quality and safety of different species of plant spagyric medicines,this experimental quality assessment protocol was developed to standardize theanalysis of spagyric plant medicines. A control and a spagyric tincture of Thujooccidentalis leaf material (NST and ST respectively) were generatedin lab and chemically interrogated via ICP-MS and GC-MS. Quantitative analysiswas employed on a principle mineral constituent, potassium (K), to determinenutritional value. Elemental complexity was also observed qualitatively. Thisexperiment further quantified the principle specialized metabolite, thujone,via GC-MS to determine any effects of the salt addition upon thujoneconcentrations. Our results indicate the ST method has a significant differencein potassium content (~145% increase), with equal elemental complexity. Astatistically significant difference in thujone ratios was also observed withinthe ST (~54% decrease). These results suggest Thujo occidentlais STcontain significant mineral/essential nutritional content which contributes tothe bioactive constituency and therapeutic value of ST’s, further, the additionof mineral salts caused the ST to undergo intra-tincture reactions leading toan alternative secondary metabolite profile compared to conventional tincturingmethods. This experiment also represents a blueprint for an all-inclusiveprotocol for the quality assessment of spagyric plant medicines which should beused in future characterizations of STs.
Keywords: Spagyric, tincture; Thujooccidentalis; thujone, potassium, nutritional assessment, qualityassessment, minerals
1.Introduction
Spagyric medicine as it has been describedby Paracelsian era alchemists and modern herbalists, is a form ofmedicinal preparation that incorporates plants, animals and minerals whichemploy the practice of “Spagyria”. This term means “to separate” (spao) and“to recombine” (ageiro). Plant spagyrics are prepared from a tripartiteextraction yielding distilled plant essential oils, alcohol fermentations,distillations or macerations, and crystallized mineral salts derived ofcalcinations [1].
The combination of this tripartite constituencyderived from the whole-plant material into a singular solution is oftensuggested to have novel phytochemical and pharmacological actions due to theentourage of phytocompounds present in a ST. To date, the literature indicatesa single quality assessment of a ST performed on Harpagophytum procumbens [2].Said paper performed a determination on the antioxidizing powers ofcommercially sourced spagyric and NSTs of devils claw. Said experiment lackedan assessment on the alkali salt constituency and any inferences aboutpotential intra-tincture reactions resultant form the combination of plantessential oils, alcohols, and mineral salts, which this paper is providing aframework for.
It was the world-renowned biochemist, Dr. LinusPauling, who suggested a form of nutritional therapy coined “orthomoleculartherapy” in his book “How to Live Longer and Feel Better”, which stated that avariety of prevalent 1st world diseases are directly linked withnutritional deficiencies. He further suggested that homeostasis is maintainedby the acquisition of primary inorganic metabolic nutrients, namely, essentialminerals, vitamins, carbohydrates, proteins and fats (including essential aminoacids) which are not endogenously produced by the metabolism [3].
It has been known for the better part of a centurythat the majority of arable farmland in the United States and subsequently theproduce grown from such land, and further subsequently the populationsconsuming the food from these farms (Participants of the Standard AmericanDiet) are severely deficient in mineral content. A congressional committee inJune 1936 addressed this fact stating that “99% of the American people aredeficient in the essential minerals.” “The alarming fact is that foods (fruits,vegetables and grains) now being raised on millions of acres of land that nolonger contain enough of certain minerals are starving us - no matter how muchof them we eat. No man of today can eat enough fruits and vegetables to supplyhis system with the minerals he requires for perfect health because his stomachisn't big enough to hold them” [4].
In the case of potassium (the mineral quantized inthis experiment), a mineral that is quintessential for human health andhomeostasis, is associated with many physiologic and pathophysiologicprocesses. Conventional scientific research has shown that upwards of 98% ofAmericans are deficient in this macro nutrient alone [21]. One study suggestedthis is so because the “world population is tending to divide itself into theunderfed in the developing countries and the badly fed in industrializedcountries.” Considering the tactics of many tyrannical authoritativeinstitutions such as the Nazi regime and cult like organizations whichintentionally malnourished their populations to make docile obedient citizensand prisoners, nutritionally related health defects are one of the greatestthreats facing the wellbeing of organized society.
Potassiumplays an essential role in in the human (and plant) primary metabolism and isprincipally involved in acid-base regulation by serving as an electrolyte [5].Potassium is a principle intracellular cation which mediates membranepotentials and supports nerve and muscle cell electrical excitation [5] [6].Potassium deficiencies have been documented as an alarmingly rising trend in the USpopulation across all age groups (12-80 years old) between 1999 and 2016. Thesedeficiencies have lead to the prevalence of hypokalemia in the affected groups[7]. Hypokalemia has a variety of symptoms that significantly reduce thequality of life of the affected person, namely neuromuscular weakness,gastrointestinal disruption, and renal dysregulation. More so, potassiumdeficiencies “have been linked in clinical studies to an increased frequency inserious arrhythmias and mortality in acute myocardial infarction” [8]. Thesestudies have shown emphatically the significance of potassium intake for propermetabolic functioning, maintaining homeostasis and a higher quality of life[9].
Plants likewise contain a diverse profile of uniquespecialized metabolites that are defined by the genetic phenotype. Specializedplant metabolites represent the dominant repository of medicinal compounds thatare further used as chemical scaffolds in the development of western drug-basedmedicines, only 36% of medicinal drugs are entirely synthetic, the other 64%are directly from or inspired by nature [22]. One such example that wasanalyzed in this study is thujone, a bicyclic monoterpenoid ketone that is theprinciple component in Thujo occidentalis. Thujone expresses canonicalbioactivity in its dose dependent actions, toxic in high doses and medicinal insmaller doses. It is a commonly occurring food and drink additive as well aspresent in multiple forms of plant medicines and preparations ranging acrossmany different genera. It first gained notoriety through the suggestion that itcaused hallucinations when ingested in the form of the popular alcoholic drink,Absinthe, however this hypothesis has since been rejected and it was determinedto be a deliriant and convulsant whichlater termed said hallucinations as “Absinthism” resultant from high alcoholcontent and intake [23]. Thujone itself acts on the 5HT3 serotonergic receptorreducing its activity, it is agonistic to the GABA receptors, and has weakaffinity for cannabinoid receptors and is suggested to be psychotropic [13,14]. The FDA and TTB have put regulations on thujone containing concoctions dueto its deliriating qualities which is currently set at 10 ppm [11], however theaforementioned study [10] found neurotoxic affects at 5 ppm thujone, half ofthe current limits, suggesting further reconsideration into its regulation andthujone containing products.
Thujones medicinalvalue has come under considerable scrutiny in recent years. Its medicinalquality has been studied in extracts of Wormwood (Artemisia absinthium)which contained high amounts of alpha and beta thujone and were shown inclinical trials to suppress tumor necrosis factor and accelerate healing inpatients with Crohn’s disease [10]. Mainly based on in vitro experiments,thujone displays a syzygy of bioactive effects: genotoxic, carcinogenic, andconvulsant on one hand, and simultaneously antimutagenic, immunomodulatory,antidiabetic with antimicrobial properties on the other [15]. The fine line between toxic and medicinaleffects in the case of thujone demands further enquiry into thujone containingconcoctions to determine the safety and optimal applications of thujonecontaining preparations.
Thujo occidentalis itself hasbeen used for centuries in homeopathic and evidence-based phytotherapies.Today, it is mainly used in homeopathy as mother tincture or dilution withother synergetic herbs such as Echinacea purpurea, Echinacea pallida andBaptisia tinctorial [16]. It has been used allopathically in clinicalevidence-based phytotherapy for respiratory infections, as an adjuvant toantibiotics in severe bacterial infections, to treat the common cold, as ananti-tumor and anti-rheumatic medicine and it also has served as a moodenhancer. Its immunopharmacological potential works by stimulating cytokine andantibody production and through activation of macrophages and otherimmunocompetent cells [16]. Other studies have shown its pro-apoptoptic andanti-angiogenic effects [16]. In summation, it can be anti-cancer, anti-tumor,anti-viral and mood elevating at lower doses, to neurotoxic, deliriating, andpossibly psychoactive at higher doses and it is still under study for thedevelopment of new medicines. Manyclinical studies have been performed on Thujo occ. containing extractswhich fail to reject its therapeutic efficacy and safety in its prevailingapplication for the treatment of respiratory infections and immunostimulatingprophylactic uses [16].
This experiment aims to provide a more inclusive chemicaland elemental fingerprinting of spagyric tinctures that includes the signatureaspect of traditional spagyric tincturing, I.e. the inclusion of plant saltsinto tinctured plants. Chemical fingerprinting in the analytical control ofherbal remedies is a widely accepted official method for determining quality ofherbal preparations [17, 18, 19]. Chemical markers chosen for this experimentwere based on the principle primary and secondary metabolites of the selectedplant material (Thujo occidentalis). Potassium being the second mostdominant mineral salt present in Thujo occ., as reported by a previousnutritional assessment on three occidentalis trees [24] and thujone,named after the genus Thuja, being the principle specialized metaboliteof Thuja occidentalis [30].
Results described here indicated significant differences inpotassium content between the ST and NST. Further analysis revealedstatistically significant results were determined in the differences of thujoneconcentrations. These results suggest a level of intra-tincture chemicalinteractions between the entourage of extracted salts and the principlespecialized metabolite thujone. However, these results should only beinterpreted for spagyric tinctures of Thujo occidentalis and thequantified metabolite thujone. Further research needs to be done on otherprimary and secondary metabolites, other research on species, and more obtusechemical fingerprints should be performed onspagyric plant tinctures to determine further possible interactionsand/or novel products formed between the large array of specialized metabolitespresent in plants and the diverse salt content of spagyric tinctures. This isespecially so in essential oil rich species, as the interactions between acidsand bases (such as the alkali salts and raw typically acidic greasyphytocompounds) can produce novel products when combined, notably so in thepresence of high proofed alcohol. It is known that the chemistry of combiningalcohols, oils and salts can easily lead to saponification reactions which candrastically alter the bioactivity of a given specialized metabolite. Although,we don’t expect this to be the case in thujone, due to the absence of carboxylgroups on thujone limiting the production of esters. The evidence providedherein argues that the case for novel product formation resultant from thespagyric tincturing method should be thoroughly investigated for more accuratechemical characterizations, drug formation identification, and for qualityassurance standards to be upheld.
2. Results
2.1PH Analysis
Table 1
Samples
Power of Hydrogen
NST (control)
5.611
ST (Treatment)
6.154
Theresults presented in Table 1, determined from 5x replicated and averaged pHmeasurements indicate a significant change in the Power of Hydrogen between thetreatment and control tinctures. These results could be a catalyst to changesin the specialized metabolic profiles. The spagyric tincturing process, in thecase of Thujo occidnetalis suggests a more alkaline final tinctureproduct, which could have implications for other compounds within thespecialized metabolic profile of T. occidentalis. Thujone pKa reported at –7.4 [20] suggests itmay have undergone a fractional deprotonation. Metabolites other than thujonecould have similar pH dependent reactions and likewise other species ofspagyric tinctures may present similar chemical reactions in relation to thealkaline salt addition, however thujone was the focus of this research and acomplete chemical finger-print was outside the scope of this study.
2.2 Salt Analysis
As shown in Figure 1, the two lab generated tinctures,tested in triplicate showed a significant compositional difference in potassiumcontent as revealed by ICP-MS quantification. Figure 3 shows a table ofelemental complexity qualitatively determined by ICP-MS. Sample preparationprocedures required a 10-fold dilution of the ST in order to achieveconcentrations within the calibration curve of the potassium standards, thissuggests the qualitatively determined elements are in a similar distribution. Theseresults are consistent with our hypothesis, failing to reject the notion thatmineral salt content would be substantially greater in a ST than a NST. This isthe first time an elemental analysis has been performed on a ST and suggeststhat further quantification of elemental constituents should be investigated soas to determine accurate mineral assessments and nutritional potency ofspagyrically tinctured plants. Our calculations suggest the spagyric tincturingmethodology yielded 0.11248 mg of potassium per 9.7184 g of spagyricallyextracted Thujo occ. leaf material.
Figure 1 Represents the ICP-MS quantitative analysis of potassium in NST and STrespectively with associated standard error of 3.79 and 2.404 PPB.
Figure 2. ICP-MS printout indicating quantified potassiumconcentrations in PPB in ST and NST (control) of Thujo occidentalis witha DBN analysis (top) and DBG analysis (middle) both displaying significantdifferences between quantity of potassium, 239.33 PPB average potassium in theST and 462 PPB in the NST from the DBG analysis (dilution scheme unaccountedfor). Bottom table indicates calibration levels employing Yttrium as aninternal standard.
Figure 3. Displays the ICP-MS printout of the blank, control (NST), andexperimental treatment (ST) indicating elemental complexity from the ST isequal to or greater than control tinctures (10x ST dilution not accounted forin table). All elements in ST are 10x greater quantity due to dilution schemespresent in sample preparation.
2.2.2.Potassium Statistical Analysis
LOD PPB
LOQ PPB
0.002858
0.00866
Table1. Limit of Detection and Limit of Quantification. The Limit of Detection (LOD)was determined to 0.002858 PPB by the conventional calculation of STDEV*10.Limit of Quantification (LOQ) was 0.00866 PPB, both suggesting therepresentative data is well within limits of the analysis.
Table 2 Figures of Merit
2.3 Thujone Analysis
Resultsdepicted in Figure 4 indicate a significant difference in thujoneconcentrations between the NST (control) and ST with ~54% difference inconcentrations, favoring the control.
Figure 4. Thujone concentrations in ST and NSTof Thujo occidentalis. Average PPM of thujone in ST = 0.153257 PPM and0.321343 PPM NST. Standard error in control = 0.0203 PPM and 0.0089 PPM in ST.
Figure 5. Chromatogram of 100mg/mL Standard concentration of Thujone with peakappearing at 6.030 minutes
Figure 6. SIM mass spectra of the indicated standard peak displaying base peakparent ion of 152.25 m/z.
Figure 7. Scan mass spectra of indicated peak showing target ion at 81 m/z withrespective fragment ions, 41, 95, 110, and 152 m/z.
Figure 8. Upper: Control tincture chromatogram displaying TIC and MIC withthujone peak indicated at 6.060 minutes. Lower: Zoomed in on TIC only ofControl tincture chromatogram with thujone peak indicated at 6.060 minutes.
Figure 9. Mass spectral SIM of the above indicated peak with a parent iondisplaying base peak at 152.25 m/z
Figure 10. Mass spectral SCAN of indicated peak displaying target ion 81 m/zand respective fragment ions of 41, 95, 110, and 152 m/z
Figure 11. Upper: TIC and MIC chromatogram of spagyrictincture sample with thujone peak indicated at 6.062 minutes. Lower: Zoomed inTIC only chromatogram of spagyric tincture sample with thujone peak indicatedat 6.062 minutes.
Figure 12. SIM mass specra of the indicated thujone peak with base peak parention at 152.25 m/z
Figure 13. SCAN mass spectra of the indicated peak displaying base peak targetparent ion at 82 m/z and respective fragment ions 41, 95, 110, 152 m/z peaks.
Area Counts m/z
Average m/z
Amount Thujone in PPM
Average PPM
Precision
Sensitivity
ST
35,493
39,416
0.138
0.153257
0.015407
1.36207
39,339
0.15296
43,417
0.16881
NST
84,216
82,645
0.32745
0.321343
0.03517
0.59666
90,803
0.35306
72.917
0.28352
Table 3 Thujone Figures of Merit
Sandard Deviation PPM
LOD PPM
LOQ PPM
36.1010
0.03502
0.10615
Table4 LOD and LOQ
2.4.Equations
Example equation for mg of K in whole treatment extract:
(Reportedaverage value of K in spagyric sample from table * dilution factor of 10) = Average PPB of K in sample
239.33* 10 = 2393.33 PPB of K
AverageK PPB in sample * 1.0E-6 = Average K mg/mL
2393.33* 1.0E-6 = 0.002393 mg of K/mL of extract
mg/mLof K in extract * total number of mL in extract = total mg K in whole extract
0.002393mg/mL* 47mL = 0.112471 mg in whole spagyric sample extract
*Thecontrol was not diluted 10x thus there is no correction for its reportedvalues. All other sample preparations were the same between the two.
3. Discussion
Based on this authors literature review, there hasonly been a single study done on the contents of a spagyric tincture [2]. Thisstudy provided evidence for long term stability of a spagyric tincture of H.procumbens active principle metabolites harpagoside, harpagide andacetoside. Said study analyzed the antioxidizing capacity of the H.procumbens tinctures for the first time further indicating STs maintaingood biological activity for at least four years post production.
Spagyria isan old alchemical medicinal procedure that is invented and described byParacelsus in his medical treatise Opus Paramirum and De NaturaRerum. Paracelsus was the first to introduce the idea of a “salt principle”to the alchemical trinity originally strictly composed of “mercury” and“sulfur”, which are philosophical metaphors used to describe qualities andattributes of nature rather than actual chemical terms. His work De NaturaRerum (On the Nature of Things) describes the importance of the “saltcomponent” in plant and mineral medicines, which contributed to theprotochemistry field of iatrochemistry and defined the distinct spagyricmedicines. Paracelsus made many contributions to medicine, such as suggestingthe dose makes the medicine and that a toxin can often be used as an antidotegiven its proper preparation [31]. He is often referred to as the ‘Father ofPharmacology’. He was further was noted for developing many mineral basedmedicines from the likes of led, tin, and copper that may have ultimately ledto his death, possibly by led poisoning.
The lack of analysis on the key signatureconstituent of spagyric medicines, I.e. salts, in the previous study demandedit be the focal point of this experiment. An ICP-MS analysis was performed on alab prepared spagyric tincture which determined a 145.6% difference ofpotassium in the spagyric tincture over the non-spagyric tincture. Theelemental complexity in ST were equal to that of the NST although we can assumethe qualitatively detected metals were similarly distributed in concentrations,although further determinations must be undertaken.
These enhanced mineral concentrations in spagyrictinctures are significant due to the fact that the majority of foodstuffscreated in the first world, and the populations consuming them, are mineraldeficient. Dr. Linus Pauling was one of the first to suggest the importance ofminerals and other essential nutrients as a quintessential tenet of health andvitality. Nutritional deficiencies are key factors in many physiological andsocial disorders prevalent in the world today [32] [33]. As previously stated,potassium, a key macronutrient, has been found to be of inadequate supplyacross the majority of arable farmland and upwards of 95% of American citizensalone. This study provided evidence that spagyric tincturing methodology can bea viable alternative medicine that provides nutritional supplementation.
Due to the nature of plants as complex mixtures ofchemicals, and tincturing methods being generally non-specific to drugselection and typically including an entourage of phytocompounds in thetinctured products, this experiment further quantized the principle specializedmetabolite of Thuja occidentalis, thujone.
Our inquiry was based on the premise that spagyricmedicine insinuates the inclusion of essential oils, salts, and alcohol in oneunified essence and this premise was fortified by observations of changes incolor, pH, and consistency of spagyric tinctures. Thus our hypothesissuggested that the alkali mineral salt addition into the plant tincture couldlead to any number of intra-tincture reactions, thus altering the quantifiableamount of principle metabolites.
We qualitatively observed a color differencebetween the ST and NST and a significant change in pH from 5.6 to 6.1 after thesalt addition. Based on these observations, we quantized thujone to determineany change in concentration that would be indicative of intra-tincturereactions between the salts and thujone.
We determined ~53% difference in concentrationsbetween the ST and NST thujone concentrations thus failing to reject the nullhypothesis of an alternative chemical profile of the spagyric tincture over thecontrol. These findings suggest intra-tincture reactions are occurring withinthe ST of T. occidentalis. These results have provided empiricalevidence that spagyric tincturing methodology can alter the principle chemicalconstituency of plant tinctures. These reactions can potentially lead toa xenobiotic chemical profile through the reaction of plant essential oils,salts and alcohols possibly leading to the formation of novel natural productsand alternative pharmacologic profiles. These results further suggest asignificant nutritional constituency that is a vector of bioactive therapeuticcomponents present in spagyric tinctures that is not typically observed innon-spagyric tinctures.
However it should be noted, these results can onlybe indicative for the spagyric tincture of Thuja occidenatlis leaves andfurther characterization should be completed on other species of spagyrictinctures to assure the quality, efficacy and nutritional content of spagyrictinctures. We further call for future research to be done on more species ofspagyric plant tinctures and more comprehensive fingerprinting to be done so asto determine the potential novel pharmacological agents occurring as a result ofspagyric plant tincturing so that the medicinal efficacy and quality of herbalmedicines can be appropriately assured.
4. Materialsand Methods
4.1Solvents and Standards
All solventsand standards (GC grade MeOH and 95% ethanol, thujone, potassium) were ofanalytical grade and were obtained from Sigma-Aldrich, US.
4.1.1Potassium Standard
A. A 1000ppmpotassium standard solution was supplied. Standard was diluted to 1ppm to serveas a 1000ppb working stock.
B. Prepare thefollowing potassium standard using the concentration volume equation C1 * V1 =C2 * V2 at the following concentrations:20, 50, 100 and 200, and 1000 μg L−1.
4.1.2Thujone Standard
A. With a 98% neat thujone standard mixture fromsigma-aldrich, dilute 110x to reach a 960ppm stock solution equivalent to958.6mg/L.
B. Prepare the following thujone standards atconcentrations of 50 mg/L, 10 mg/L, 5 mg/L, 1 mg/L, and 0.5 mg/L.
4.2 Sampleallocation
Thujooccidentalis leaves were harvestedfrom multiple cedar trees around Harlow lake in northern Michigan in lateAugust (coordinates: 46.62866° N, 87.48971° W). The fresh leaves wereimmediately stored in -20ºC freezer for 14 days before experiment began.Samples were identified as Thujo occidentalis by botanists with NorthernMichigan University.
4.3 Controltincture (NST) preparation
A. Wearinggloves to avoid contamination, homogenize the Thuja occidentalis sampleinto a fine powder.
B. Mass out5-10 grams of Thuja occidentalis sample into a beaker and record andlabel accordingly.
C. Using a20:1 volume mL/weight g solvent to herb material ratio, pour solvent over Thujaoccidentalis samples and seal the top with parafilm.
*For example, if 5 grams of occidnetalissample was measured in beaker, add 100mL of 95% EtOH solvent, if 6 grams, at120 mL, etc.*
D. Assemblethe Soxhlet apparatus.
E. Poursolvent with plant material into the thimble, allowing for the siphon to prime.*Add filter at the bottom of the thimble.*
F. Turn onheat so that solvent begins distilling at 78.2ºC. Run Soxhlet until the solventruns clear through the siphon.
G. Whensolvent runs clear, remove the plant matter, disassemble the apparatus, place afunnel on top of the boiling flask containing the extract and press the plantmatter through a 3 micron filter to reclaim any left-over solvent absorbed inthe plant matrix.
H. Filter theextract and separate it into two equal fractions, label one control and theother spagyric.
4.4Spagyric tincture (ST) preparation
A. Take theremaining solid plant matter and place it into a crucible to begincalcination.
(Steps B, C,and D can also be accomplished with a muffle furnace set to 450ºC for 16 hours,following the protocols at [25].)
B. Calcine thematerial between 260-550ºC until ash turns pure white.
C. Remove theplant material/ash from the oven and crucible after about 6 hours and grind theplant matter with a mortar and pestle to encourage calcination.
D. Placematerial back into crucible then calcine again until plant material turns purewhite, indicating absence of carbon and total calcination.
E. Place whitecalcined ash into small glass dish and pour water ~3 times its volume over theash in order to dissolve the calcined material.
F. Filter theinsoluble ash out with a three micron filter.
G. Slowlydissolve the filtered aqueous ash solution in to a small dish to crystallizethe remaining salts.
G. Slowlydissolve water until crystalline salts appear, collect and pulverize them to afine powder in a mortar and pestle, being careful not to spill any.
H. Add thepurified and crushed mineral salts into the fraction labeled “spagyric”.
I. Add the“spagyric” to a reflux apparatus and reflux the spagyric solution for ~4 hourson low heat (~65ºC). This encourages total dissolution of salts into solutionand follows the commonly accepted and traditionally prepared method.
J. Remove thespagyric tincture from the reflux apparatus and put it in an amber vessel intoa refrigerated environment away from direct light. Label and date until theyare ready for sample preparation and analysis.
4.5 GC-MSSample Preparation and Analysis Conditions
A. Remove thecontrol and spagyric tinctures from storage and follow the below steps,repeating twice for processing both the control and the spagyric.
B. Ascertainsix 2mL vials and annotate their weight. Use three replicates of each sample.
C. Remove 1 mLof the NST and ST and place into the vial. Using a speed vacuum, reclaim theethanol extracting solvent, removing the solvent from the tinctures leaving thecrude extract
D. Weigh andrecord the crude extract residue of both ST and NST vials, record exact weightof crude extracts.
E. Add 1mL ofGC grade MeOH to crude extract samples and shake until fully dissolved, thisstep may require sonication. Transfer 1mL of the sample into a GC vial.
F. Centrifugesamples at 15,000 RPM for 10 minutes, discard pellet.
G. Filterthrough 0.45 micron syringe filter.
4.5.1 GC-MSConditions
A. Prepare theGC-MS instrument conditions and gradient scheme according to these settingsadapted from [26]: Employ a 30m long capillary column with 0.25mm innerdiameter and 0.25um film thickness. A 10:1 split ratio and 1uL injectionvolume, Nitrogen carrier gas, with 1.5mL/min flow rate, injector temperatureset to 300ºC and initial oven temperature of 70ºC, held for 3 min, increased at20ºC.min to 190ºC held for two minutes. MS will be set on EI (electronionization mode) with ion source temperature at 250ºC. Mass spectra scanningrange from 35 – 360 m/z.
B. Prepare ablank, collect the standards and the control and spagyric tincture samples andthen inject the samples into the GC in triplicate.
4.6 ICP-MSSample Preparation and Analysis Conditions
4.6.1 Microwave Digestion:
A. Take 3 mL of the tincture and add it directly toa digestion vessel.
B. Add 5 mL of65% HNO3 and 2 mL of 30%H2O2.
C. Coversamples and place into microwave digestor. Set digestion program at 1 min at250 W, 1 min at 0 W, 5 min at 250 W, 5 min at 400 W, and 5 min at 600 W.
D. Aftercooling, transfer digested samples to volumetric flask and dilute to 50 mLusing de ionized water and finally transfer to a 50 mL polyethylene flask.
4.6.2 ICP-MS Conditions
A. Set theICPMS to the following conditions: (ICP-MS, 7900, Agilent) instrument with the followingcondition/parameters; Nebulizer gas-flow: ∼1 L min−1, Auxiliary gas-flow: ∼1 Lmin−1, Plasma gas-flow: ∼15 L min−1, Helium (He) gas-flow in Reaction Cell:∼0.2 mL min−1, Reflected power: ∼45 W, Forward power: ∼1500 W, Analyzer vacuum:∼6 × 10-5, Detector: EM, Replicates: 3, Sweeps/replicate: 100.
B. Inject thecontrol (NST) and experimental spagyric tincture (ST), blanks, and standardsinto the ICPMS.
4. Validation
4.1Potassium Validation
Preparation of the control and spagyric tincturesfor ICP-MS mineral analysis of the element potassium are be based on the workof Ilyas Ahmad et al. [28]. Using a potassium Standard at five differentconcentrations (20, 100, 250, 500, and 1000 μg L−1). Both tincture samples willbe prepared via microwave digestion according to the description in the methodssection 4.6.1. Potassium content is quantitively determined from both controland spagyric tincture samples using an inductively coupled plasma mass spectrometry(ICP-MS, 7900, Agilent) instrument programmed with the parameters described inthe ICP-MS Conditions Methods section 4.6.2
The quantification of potassium was made by ICP-MSagainst a calibration curve obtained with potassium at six differentconcentrations in the linear range of 20-1000 µg/mL. The correlationcoefficient (r2) of the standard curve in the linear plot of log-transformeddata was r2 = 0.99986 (y = 0.0114371*x − 161.22955), indicating good linearitybetween the log-transformed areas and log-transformed concentrations within thetested concentration range.
The limit of detection (LoD) and limit ofquantification (LoQ) were calculated according to conventional guidelines [27]on the basis of signal-to-noise ratios (S/Ns) of 3:1 and 10:1, respectively, byinjecting a series of dilute solutions of known concentrations of potassium inthe aforementioned range. The LoD for potassium in the adopted analyticalconditions was 0.002858 µg/mL, while the LoQ was 0.00866 µg/mL.
Figure 14 displays ICP-MS DBNcalibration levels, blank, and line of best fit.
Figure 15 displays ICP-MS DBG calibration levels, blank, andline of best fit.
4.2 Thujone Validation
Specific parameters for the chromatographicseparation and mass spec. are given in Alshishani et al. [26]. employing a 30mlong capillary column with 0.25mm inner diameter and 0.25um film thickness. A10:1 split ratio and 1uL injection volume, Nitrogen carrier gas, with 1.5mL/minflow rate, injector temperature set to 300C and initial oven temperature of70C, held for 3 min, increased at 20C.min to 190C held for two minutes. MS willbe set on EI (electron ionization mode) with ion source temperature at 250C.Mass spectra scanning range from 35 – 360 m/z. Gas chromatography was chosenbecause of the analyte being volatile and stable at high temperatures capableof withstanding the high temperatures of GC (only degrading at 203 degreesCelsius).
The quantification of thujone was made by GC-MSagainst a calibration curve obtained with thujone at five differentconcentrations in the linear range of 0.5-100 mg/L based on the work of ChajdukM. et al. and the NIST Standard Mass Spectral Database [29] where parent ionand ion fragments were identified as 152, 110, 95, 81 and 41 m/z respectively.The correlation coefficient (r2) of the standard curve in the linear plot oflog-transformed data was r2 = 0.9993 (y = 3401.9x-2659.7), indicating good linearitybetween the log-transformed areas and log-transformed concentrations within thetested concentration range.
The limit of detection (LoD) and limit ofquantification (LoQ) were calculated according to conventional guidelines [27]on the basis of signal-to-noise ratios (S/Ns) of 3:1 and 10:1, respectively, byinjecting a series of dilute solutions of known concentrations of thujone inthe aforementioned range. The LoD for thujone in the adopted analyticalconditions was 0.03502 m/z area counts, while the LoQ was 0572.756 m/z areacounts.
Figure 1. Standard Curve displaying 0.5, 1, 5, 10, and 50mg/L of Thujone standards. R-squared of 0.9993 and y = 3401.9x - 2659.7
4.3 Statistical Analysis
4.3.1 Thujone Statistical Analysis
The statistical analysis of the quantitativeresults for each analysis was determined with ANOVA single factor testprocessed with Microsoft Office Excel 2017. For the thujone analysis, an alphavalue at (0.05), F-value = 57.491, F-critical value = 7.709, and a P-value =0.0162, thus rejecting the null hypothesis and indicating significantdifference between concentrations of thujone between ST and NST.
4.3.2 Potassium Statistical Analysis
For the potassium analysis, an alpha value at(0.05), F-value = 6299.58, F-critical value = 7.709, and a P-value = 1.51E-07,thus rejecting the null hypothesis and indicating significant differencebetween concentrations of potassium between ST and NST.
Author Contributions: This researchreceived no external authorships.
Funding: “This research received noexternal funding”
Data Availability Statement: Originalexperimental data is available at the following google document link:https://drive.google.com/drive/folders/1VpM-kF6avJl2t4mpbZKS6HCijSbuaUVv?usp=sharing
Acknowledgments: The author would liketo thank the faculty and staff at Northern Michigan University for fosteringhis education. He would further like to thank all peers who encouraged andtaught him along his scientific and alchemical journey.
Conflicts of Interest: “The authors declare no conflict of inter
Sample Availability: Samples of thecompounds are available from the author.
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