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Analysis of Iohexol using the Advion Interchim Scientific AVANT™ HPLC and expression® CMS System

Introduction

Iohexol is a widely used non-ionic imaging agent that improves contrast for x–ray analysis. Its low osmolality allows for a rapid clearance via the kidney, preventing reabsorption and further metabolization[1]. This makes iohexol a compound with a better safety profile compared to other imaging agents[2]. 

In many clinical imaging applications, imaging agents/contrast agents are administered to patients to improve the contrast and spatial resolution of the scan. Due to toxicity and side effects of imaging agent, preparations of their known concentrations and their purity analysis are very important for their safe use and accurate diagnostic. 

In this application note, a simple and accurate HPLC-CMS method for iohexol analysis is introduced.

Method

Method Setup

All solvents used in the application were HPLC grade.

Two Iohexol standards were obtained from Sigma Aldrich.

One was a certified reference material with purity of 99.99%, the second one had a stated purity of ≥ 95%.

All experiments were performed on an Advion Interchim Scientific expression® Compact Mass Spectrometer coupled with an AVANT™ UHPLC system with parameters as shown in Table 1.

Table 1: HPLC/MS Method

HPLC/UV/MS Analysis of Iohexol

At room temperature, iohexol will isomerize with two peaks detected in its HPLC/UV/MS analysis. 

These two peaks (Figure 1A) come from hindered rotation of the anilide N-acetyl group due to the bulky iodine atoms attached to the central benzene ring of the iohexol. Those two compounds are essentially “rotational isomers” that interchange slowly at room temperature in aqueous solution.

Both peaks are confirmed with MS analysis to show the same m/z at 821.9 (Figure 1B and 1C) and no difference was visible in their in-source CID mass spectra (Figure 2A and 2B). 

Since both rotamers contribute to the compounds toxicity and imaging enhancing capabilities in x-ray analysis, the sum of both peaks will be used for any further iohexol analysis in this application note. 


Figure 1: (A) HPLC chromatogram (254 nm) of iohexol, (B) The extraction ion chromatogram of protonated iohexol at m/z 821.8, (C) The averaged MS spectra from peak at RT 4.06 min.


Figure 2: (A) The averaged in-source CID mass spectra from peak at RT 3.61 min, (B) The averaged in-source CID mass spectra from peak at RT 4.06 min.

Purity Determination by HPLC/UV Analysis

By comparing the HPLC response from iohexol sample to the response of the iohexol certified standard at a similar concentration, the peak area ratio of sample to certified standard can provide a quick concentration and purity analysis. 

The equation to calculate the concentration ratio of iohexol is shown below:

The HPLC chromatograms of iohexol sample and certified reference are shown in Figure 3A and 3B.

The averaged value of summed two peak areas is 714 for iohexol sample (Figure 3A), and 740 for iohexol reference standard (Figure 3B). With the calculation of concentration ration, the calculated concentration of iohexol in the sample is 0.0964 mg/ml which equals a purity of 96.4% – in line with the stated product purity of ≥ 95%.


Figure 3: (A) HPLC chromatogram (254 nm) of iohexol sample (0.1 mg/ml) (B) HPLC chromatogram (254 nm) of iohexol reference standard (0.1 mg/ml).

Quantitation by HPLC/UV Analysis

To check the purity of an iohexol sample more accurately, a calibration curve of iohexol certified standard material was created with five different dilution levels from 25 to 500 μg/mL and with triplicate injections for each concentration. The R-squared value of the resulting linear calibration function is 0.9999 (Figure 4) showing excellent linearity. 

By way of the iohexol calibration curve approach, the purity of the iohexol sample was determined to be 97.5%. 

This value is also right above the stated purity of min 95% of the sample and differs by only 1.1% from the measured value by direct UV response ratio analysis of the iohexol sample to iohexol reference standard.

Both purity determination by way of a calibration function or by direct UV response ratio analysis can be used for organic chemicals with UV absorbances if certified reference standard material is available.

The calibration function method will provide a more accurate measurement.


Figure 4: Calibration curve of iohexol by HPLC chromatogram (254 nm)

Conclusion

The Advion Interchim Scientific AVANT™ (U)HPLC system can provide accurate chromatographic methods for the purity analysis of imaging agents as shown for iohexol. Coupling UHPLC with the Advion Interchim Scientific expression® Compact Mass Spectrometer not only provides confirmations of target compounds via their mass and in-source fragmentation pattern, but also allows for rapid determination of impurities. 

REFERENCES
[1] T. Almen, Development of nonionic contrast media., Invest. Radiol. (1985) Investigative Radiology. 1985, 20(1), S2-S9.
[2] R.D. Moore, E.P. Steinberg, N.R. Powe, R.I. White, J.A. Brinker, E.K. Fishman, S.J. Zinreich, C.R. Smith, Frequency and determinants of adverse reactions induced by high-osmolality contrast media., Radiology. 1989, 170, 727-32.

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Extraction and Purification of 3 Curcuminoids from Turmeric Powder

Instrumentation:
Flash: puriFlash® XS520
TLC: Plate Express™ TLC Plate Reader
Mass Spec: expression® Compact Mass Spectrometer
Sampling: ASAP® Direct Analysis Probe

Introduction

Curcuminoids are natural polyphenol compounds derived from turmeric root (Curcuma longa). They are reported to have antioxidant activities1. Curcumin is the main curcuminoid found in turmeric. It is commonly used as an ingredient in dietary supplements and cosmetics, flavoring in culinary dishes, and a yellow-orange food coloring.

In this application note, a method to separate and purify 3 curcuminoids from turmeric powder using flash chromatography with the Advion Interchim Scientific puriFlash® XS520 Plus, TLC with mass spectrometry with the Plate Express™ TLC Plate Reader and expression® CMS is demonstrated. Fractions were identified using the Atmospheric Solids Analysis Probe (ASAP®).

Curcuminoid Extraction

The turmeric powder was weighted out (57.3 g) and transferred to a wide mouth glass bottle. Ethanol (250 mL, 200 proof) was added to the bottle and the mixture was stirred for 18 hours while covered with foil. The compounds of interest are sensitive to light. The slurry was then filtered and the filtrate was concentrated to dryness to form an amber oil (6.4 g).

Figure 1: Structures of curcuminoids.

Figure 2: Store-bought turmeric powder (left) and crude extract oil (right).

TLC/MS Analysis

The Advion Interchim Scientific Plate Express™ paired with the expression® CMS allows for easy identification of spots on TLC plates without the need for purification or sample preparation (Figure 3).

Initial TLC analysis showed 4 spots (dichloromethane:methanol, 97:3). The three lower spots were highly fluorescent, as expected for the curcuminoids of interest. TLC spots were analyzed by APCI ionization in negative ion mode. The bottom 3 spots were characterized by mass spectrometry.

Figure 3: Advion Interchim Scientific expression® CMS and Plate Express™ TLC Plate Reader (left) and close up of the TLC plate extraction head (right).

Figure 4: Developed TLC plate visualized at 365 nm. Resulting mass spectra of cur cumin (top), demethoxycurcumin (middle), and bisdemethoxycurcumin (bottom).

Flash Purification

An isocratic method was used as the separation shown on TLC was optimal as is. The crude material was purified on a 25 g, 15 μm spherical silica gel column (PF-15SIHC-F0025). A crude weight of 64 mg was dry-loaded onto 500 mg of silica gel and loaded into a 4 g dryload cartridge (PF-DLE-F0004).

Figure 5: Resulting flash chromatogram from developed TLC Plate.

Fraction Identification by ASAP®/CMS

The expression® CMS with the ASAP® Direct Analysis Probe allows for easy identification of compounds without the need for LC/MS or sample make-up.

The pure fractions (1.1, 1.3, and 1.5) were analyzed using the ASAP® probe with APCI ionization and positive polarity CMS. The curcuminoids ionize well in both APCI positive and negative polarity, however (M+H)+ ions showed less fragmentation. The detected masses are consistent with the theoretical [M+H]+ m/z values.

Figure 6: Advion Interchim Scientific ASAP® Direct Analysis Probe being inserted directly into the APCI-enabled ion source of the expression® CMS.

Figure 7: Mass spectra of fractions.

The purified fractions were concentrated to dryness to give solids I (14.1 mg), II (5.6 mg) and III (6.7 mg) respectively, which represents Curcumin (I), demethoxycurcumin (II), and bisdemethoxycurcumin (III) at 53.4%, 21.2%, and 25.3% of the isolated curcuminoid profile. These results are consistent with reported literature values2.

Confirmation of Compound Purity by RP-HPLC

Figure 8: UV Scan of purified fraction mixture.

Reverse Phase High Performance Liquid Chromatography (RP-HPLC) allows for a separate confirmation of compound purity after flash chromatography. An equal mixture of all three compounds was combined and run on a Phenomenex Kinetex® 5 μm Biphenyl 100 Å 50 x 2.1 mm column using isocratic ACN:Water (v:v, 55:45) with 0.2% formic acid. As expected, the elution order of the three curcuminoids changed order with now eluting III, II and I (Figure 8). After developing this method, the respective single collected fraction was injected and analyzed for purity and again confirmed by MS analysis.

Figure 9: UV Scan and mass spectrum of Curcumin Fraction 1.1.

Figure 10: UV Scan and mass spectrum of Curcumin Fraction 1.3.

Figure 11: UV Scan and mass spectrum of Curcumin Fraction 1.5.

Conclusion

With a combination of TLC chromatography, flash chromatography and mass spectrometry support at various stages of the process (TLC plate identification, fraction confirmation and secondary purity analysis), we can purify curcuminoids from Turmeric powder at confirmed purity levels of >95%.

References:
1Jayaprakasha et al. Antioxidant activities of curcumin, demethoxycurcumin and bisdemethoxycurcumin. Food Chemistry, Volume 98, Issue 4, 2006, Pages 720-724. ps://doi.org/10.1016/j.foodchem.2005.06.037.
2Praveen et al. Facile NMR approach for profiling curcuminoids present in turmeric, Food Chemistry, Volume 341, Part 2, 2021, 128646, https://doi.org/10.1016/j. foodchem.2020.128646.

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Soil Analysis using the Advion Interchim Scientific SOLATION® ICP-MS

Introduction

Environmental contaminants generated by human or industrial activities often find their way to the soil via runoff waters or deposition from the air. These contaminants can be taken up by plants and move up the food chain leading to potentially significant impacts on human and animal health. Therefore, it is not only important to monitor the levels of essential nutrients in the soil that are key for healthy plant growth, but it is also imperative that the levels of contaminants are monitored.

In this application note we present a method for routine analysis of 21 elements using the SOLATION® Inductively Coupled Plasma Mass Spectrometer (ICP-MS). A group of unknown soil samples and a CRM were digested using EPA 3051a and analyzed according to method 6020a requirements.

Experiment

Reagents and Materials
• Nitric acid (Aristar Plus, trace metal grade)
• Hydrochloric acid (Aristar Plus, trace metal grade)
• Water, type 1 (18.2 MΩ, Elga point of use system or equivalent)
• NIST CRM2706 “New Jersey Soil, Organics and Trace Elements”
• Spex ‘CL-ICV-1’ multi-element solution
• Aluminum standard solution (1000 μg/ml, Claritas ppt grade)

Instrumentation
1. Anton Paar Multiwave 5000 with the 20SVT50 rotor (20 position, 50mL vessels that vent at 40 bar (580 psi)
2. OKF high speed multi-function grinder
3. Advion Interchim Scientific SOLATION® ICP-MS

Standards

Calibration standards were prepared in the same acid proportions as the digested samples (9mL HNO3+ 3mL HCl, or 3:1). One liter of 3% HNO3+ 1% HCl was made as the diluent for standards, for the final dilution of samples, and to use as a calibration blank.

Standards were made using the Spex multi-element solution ‘CL-ICV-1’ and the single element Aluminum standard. Aluminum was added separately to the mix to account for the high levels of this element in soil.

Samples and Preparation

Four soil samples were dried at 60°C overnight, then finely ground using an OKF high speed multi-function grinder to make a homogeneous mixture. As per EPA method 3051a “Microwave assisted acid digestion of sediments, sludges, and soils”, 0.5 g of each sample were transferred to microwave vessels and mixed with 9mL nitric and 3mL hydrochloric acids. The vessels were then capped and run using the method outlined in Table 1. After digestion the samples were filtered, brought to volume with deionized water in a 50mL volumetric. A 1.0mL aliquot was then diluted to a final volume of 50mL with the prepared diluent for a nominal final dilution of 5,000x depending on the initial sample weight.

Table 1: Microwave Digestion Program.

For QC purposes the four unknown soil samples were prepared as a sample, duplicate, and spike. They were independently digested where the first two were used to compare the repeatability of the sample preparation, while the third one was spiked prior to the digestion to establish analyte recovery of the digestion procedure.

To verify the accuracy of the results, we included the standard reference material, NIST 2706 “New Jersey soil, organics and trace elements”, which includes certified values for all analytes reported in this study.

The samples were analyzed using a SOLATION® ICP-MS. The SOLATION® instrument configuration for this analysis was a cyclonic spray chamber with a Micromist® concentric nebulizer and a one-piece torch. Ni sampler and skimmer cones were used throughout the study. The plasma operating parameters were:

Table 2: Plasma Operating Parameters.

ICP-MS Method

Integral to the SOLATION® ICP-MS is an octupole collision cell that is used for addressing interferences from polyatomic ions, especially for the transition metal elements. It is critical for robust and routine trace element analysis that the octupole cell does not become contaminated which could cause drift and unnecessary downtime. Therefore, the ion path of the SOLATION® ICP-MS was designed to have the collision cell out of the direct line of the plasma. Ions passing through the interface are directed through a 90 ̊ turn and focused onto the entrance of the octupole using a quadrupole deflector (QD). Light and neutral particles continue through the QD and away from the cell.

The collision cell in the SOLATION® ICP-MS can be operated in “He Gas” mode in which the cell is filled with He to act as a collision gas, or in “No Gas” mode in which the cell is empty. The “He Gas” mode is used for isotopes subject to polyatomic interferences while the “No Gas” mode is used for the rest of the isotopes. The rapid switching between “He Gas” and “No Gas” modes on the SOLATION® (< 5 sec) ensures that analytical runs can be kept short, thereby improving productivity.

The helium flow used for “He Gas” mode in this application was 6 ml/min. Table 3 lists the elements used for this analysis and their isotopes, and the mode used for each.

Table 3: A list of the elements included in this study together with their isotopes and the gas mode used for the analysis.

Results and Discussion

The results summarized in Figure 1 show excellent agreement between the measured data for CRM2706 and the reported extracted levels for these elements. A slightly higher recovery was observed for K and Al, possibly due to variability in the extraction efficiency of this digestion method.

Figure 1: Certified reference material recovery data.

As shown in Table 4 spike recoveries averaged between 75% and 125% for all elements, with the exception of Al; This was likely due to the small size of the spike compared to levels of Al in the samples. Included in the same table are the results from the duplicate digestions/analyses for these elements. On average, the duplicates were less than 20% apart with most elements showing excellent repeatability of <5%.

Table 4: Average spike recoveries and duplicate repeatability for the various samples.

Summary

In this application brief we report on the analysis of trace elements in soil using the Advion Interchim Scientific SOLATION® ICP-MS. Excellent recoveries were observed for both spiked samples and CRMs. The combination of the quadrupole deflector and the collision cell minimizes drift and ensures accuracy and precision over time. The reported method benefits from the fast collision cell gas switching capabilities of the SOLATION® to analyze a wide range of elements in soil for rapid, accurate and reproducible results.

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Mass Directed Fraction Collection of Natural Products: Examples from Turmeric and Green Tea Extract

Flash: puriFlash® 5.250
Mass Spec: expression® CMS
Sampling: ASAP® probe

Introduction

Flash chromatography has traditionally used UV absorption as the main method of detection for compounds during a purification process. While UV absorption is broadly applicable to many classes of compounds, it has limited specificity to individual compounds in a mixture and misses classes of compounds that do not carry chromophores.

Mass-directed fraction collection gives users the ability to collect fractions based on mass spectrometry detection (MS) which is based on ions specific to individual compounds and provides specific molecular information. This allows for simplification in the overall purification process and greater confidence in the identity of each isolated compound.

Here we describe methods of isolating natural products from green tea and turmeric powder by mass-directed fraction collection during flash chromatography and preparative LC. For demonstration purposes, the isolated compounds were then additionally confirmed by Atmospheric Solids Analysis Probe (ASAP®) MS or HPLC-MS.

Introduction to Curcuminoids

Curcumin is the main curcuminoid found in turmeric root (Curcuma longa). It is commonly used as an ingredient in dietary supplements and cosmetics, flavoring in culinary dishes, and a yellow-orange food coloring. Curcuminoids have been reported as having antioxidant and anti- inflammatory activities.

Store-bought turmeric powder (57.3 g) was extracted in ethanol, then filtered through filter paper, and concentrated. This yielded a crude extract oil of 6.4 g containing the three curcuminoids of interest (also compare TLC analysis in Figure 4).


Figure 1: Structures of the curcuminoids of interest.


Figure 2: Store-bought turmeric powder.


Figure 3: Crude extract oil from turmeric powder.


Figure 4: TLC analysis at 365 nm of turmeric extract (97:3 DCM:MeOH) and the chromatogram of the method transferred to the puriFlash® 5.250 using UV detection.

Method Development

The turmeric extract was first analyzed on a TLC plate and then the method transferred to the puriFlash® 5.250 system using UV detection at two wavelengths. Four compounds were detected at 254 nm with three assumed curcuminoids detected at 427 nm, however, there is no specificity for the individual compounds in UV detection.

An isocratic method (97:3 dichloromethane:methanol) was used as the separation shown on TLC was optimal. The crude material was purified on a 12g, 15 μm spherical silica gel column (PF-15SIHC-F0012). A crude weight of 32 mg was dry-loaded onto 250 mg of silica gel and loaded into a 4g dryload cartridge (PF- DLE-F0004). Fractions were collected using the XIC channels for each compound of interest.


Figure 5: Screenshot of the flash chromatography method run with parameters.

The mass spectrometer settings are controlled through the InterSoft®X software on the puriFlash® system. The mass spectrometer was fitted with an APCI source and run with negative ionization acquisition mode.


Figure 6: Screenshot of the mass spectrometer parameters for chromatography run.

Experiment

Mass-Directed Fraction Collection

The extracted ion chromatogram (XIC) created by plotting the intensity of the observed signal at a chosen mass-to- charge value. This allows for a low-noise signal of compounds of interest. Here the XIC channels are set to detect the three curcuminoids of interest.


Figure 7: TLC Analysis of turmeric extract (97:3 DCM:MeOH) and chromatogram of method transferred to the puriFlash® 5.250 using MS XIC detection.




Figure 8: The mass spectra for each peak as provided by the puriFlash® InterSoft®X software.

ASAP® MS Fraction Confirmation

The pure fractions (1.1, 1.2, and combined 1.3 and 1.4) were additionally analyzed using ASAP® negative polarity MS. The detected masses are consistent with the theoretical [M-H]- m/z values.




Figure 9: The mass spectra of the isolated compounds confirming their identity and purity.

Introduction, Green Tea Catechins

Dry green tea typically consists of 10-30% of polyphenols based on dry weight with catechins being the major tea polyphenols including: (−)-epigallocatechin (EGC), (−)-epigallocatechin-3-gallate (EGCG), (−)-epicatechin- 3-gallate (ECG) and (−)-gallocatechin gallate (GCG). EGCG is the most abundant and biologically active catechin, separating and purifying catechins from raw tea extract can greatly increase their market availability and value.

Dry green tea leaves were extracted into hot water, then partitioned with ethyl acetate, filtered through filter paper, and evaporated to give a crude extract. The dry extract was then dissolved in 7.5 mL of water and filtered with 0.2 μm filter before further processing.


Figure 10: Green tea leaves steeping.


Figure 11: Major Catechins in Green Tea.

Method Development
With HPLC-UV/MS analysis, EGC, EGCG, GCG, EC and ECG are detected in the tea extract (Figure 12).

Solvent A: Water
Solvent B: Methanol
UV: 275 nm
MS: full scan from 150-900
Column: US15C18HP-250/046


Figure 12: The HPLC-UV chromatogram of green tea extract, MS data and a standard for EGCG was used for compound confirmation (data not shown).

The mass spectrometer settings are controlled through the InterSoft®X software on the puriFlash® system. The mass spectrometer was fitted with an ESI source and run with negative ionization acquisition mode.


Figure 13: Screenshot of the mass spectrometer parameters for chromatography run.

Mass-Directed Fraction Collection
Here the XIC channels are set to detect the 4 catechins of interest. EGCG and CGC are isomers and therefore share the same mass.


Figure 14: Chromatogram of the method transferred to the puriFlash® 5.250 using MS XIC detection.


Figure 15: The mass spectra for each peak as provided by the InterSoft®X software.

Conclusion

• With natural products isolation, one of the biggest challenges is the identification of compounds of interest in complex extract mixtures.
• Using MS and chromatography in tandem we can separate and identify compounds in a complex mixture with a high degree of purity and accuracy without the need for further identification of fractions collected.
• The fractions collected can be characterized directly through the MS data provided by the InterSoft®X software on the puriFlash® systems.
• The puriFlash® 5.250 and expression® CMS make a powerful duo in the purification and identification of natural products such as the catechins found in green tea and the curcuminoids found in turmeric.

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High-Throughput Purification of Five Over-the-Counter & Prescription Drug Compounds by Reverse-Phase Preparative LC-MS

Instrumentation:

puriFlash® 5.250
expression® CMS
Uptisphere® StrategyTM column US5C18HQ-150/300

Authors:

Advion Interchim Scientific, Montluçon, France Headquarters

 

Introduction

Purification is a critical step in drug development. From research, to scale-up to process, purification and confirmation are essential steps in bringing a drug to market. It is essential to have a high-throughput solution that offers sufficient quantity and reproducible quality of purified compounds. The separation of the active pharmaceutical ingredients (APIs) from their impurities can be easily achieved with a preparative chromatography system.

This application note features the purification of five active ingredients found in over-the-counter (OTC) drugs including caffeine, glafenine, ketoprofen, flavone, and fenofibrate (Figure 1), by a preparative purification workflow with confirmation using a compact mass spectrometer.

Figure 1: The five compounds of interest include caffeine, glafenine, ketoprofen, flavone, and fenofibrate. Chemical structures and pharmaceutical use cases are highlighted below.

Caffeine: A natural chemical with stimulant effects, caffeine can be found purified in tablet form, or naturally occurring in coffee, tea, cocoa and more.

Glafenine: A nonsteroidal anti-inflammatory drug (NSAID), glafenine was removed from the market in 1991 due to a high risk of anaphylaxis.

Ketoprofen: A prescription-based nonsteroidal anti- inflammatory drug (NSAID), ketoprofen is used to treat inflammation, swelling, stiffness and joint pain. The drug was discontinued in 1995 due to increased risk of heart attack, stroke, irritation and other issues.

Flavone: A metabolite and nematicide that commonly exists in plants.

Fenofibrate: A prescription medication used to reduce and treat high cholesterol and triglyceride (fat-like substances) levels in the blood.

Experiment

Exploratory LC Separation

Figure 2: To confirm the presence of the pre-identified compounds, an exploratory LC-UV run confirmed the presence of the drug compounds prior to purification.

Preparative LC Run

Following the positive ID of the five compounds of interest and their elution points, the drug mixture was then ready for a preparative LC-UV run on the puriFlash® 5.250 iELSD. The purification is aided by the iELSD pack, enabling the detection of chromophore-free compounds (Figure 3).

Results and Validation

Separation & Purification Results

The identity of the separated compounds was confirmed using the Advion Interchim Scientific expression® Compact Mass Spectrometer, quickly and accurately identifying the compounds of interest.

The purity of these compounds can be verified using analytical scale HPLC.

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SOLATION®, A New ICP-MS for the Detection of Heavy Metals in Cannabis and Hemp

Introduction

Cannabis and hemp products are becoming much more available for medicinal and recreational use making routine testing for toxic heavy metals much more important.  Advion Interchim Scientific introduces the SOLATION® ICP-MS for the analysis of heavy metals in cannabis plant and cannabis product samples.  While there are no federal guidelines for heavy metals in cannabis, states where cannabis use and production are legal have adopted exposure limits and QC criteria for Arsenic, Cadmium, Mercury, and Lead based on USP<233>.  Here, we report the results of our sample analysis using these guidelines. 

Methods

Cannabis flower was purchased locally and finely ground for analysis.  Samples are prepared using a microwave digestion system (CEM Mars 6, Matthews, NC).   Method validation for USP<233> is based on accuracy, using spike recoveries, repeatability based on the %RSD of six independently digested replicate, and ruggedness, where those 6 replicates are run a second time by another analyst, another instrument, or on another day.  The spike levels are based on the “action level” defined by the California maximum permitted daily exposure (PDE) limits as a guide:  Lead 0.5 µg/g, Arsenic and Cadmium 0.2 µg/g, and Mercury 0.1 µg/g are used to define the 100% spike level.  Samples are also spiked at 50% and 150% of the action level. 

Preliminary Data

For digestion, 0.5 g (+/- 0.002g) of sample is treated with 9mL conc. HNO3 and 1mL conc. HCl in a microwave vessel and allowed to react for 15 minutes prior to being capped.  The vessels are loaded onto the carousel in the microwave and the “one touch” cannabis method, supplied by CEM, is used.  Samples are brought to 200°C in 30 minutes, held there for 10 minutes, and allowed to cool.  The result is a clear, particle free solution. The SOLATION® ICP-MS was used to analyze the samples for As, Cd, Hg, and Pb after digestion and dilution.  The results show that the SOLATION® ICP-MS was able to produce accurate values as measured by the spike recoveries which were well within the 70-150% range.  The results from the 6 independent digests were within the defined limit of 20% RSD.  Repeat analysis of the 6 digests on a separate day showed good agreement with the initial results and were within the 25% RSD spec. defined by USP<233>.  Overall results show that the SOLATION® ICP-MS is an effective instrument for the analysis of cannabis and hemp samples.

 

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Mass Directed Fraction Collection of Natural Products: Examples from Turmeric & Green Tea Extract

Introduction

Natural products have been a source of inspiration for pre-clinical drug discovery both by exploring traditional medicines and to discover new spaces in pharmacology. Isolation and characterization of natural products remains a major barrier in drug discovery. Isolation is generally done on an analytical scale and then compounds are characterized fully before any scaleup purifications are attempted. The ability to purify compounds in complex natural product mixtures by highly specific MS data allows for the simplification of the purification and characterization steps of the process. Here, flash and prep chromatography are coupled with MS detection to purify natural products in green tea and turmeric extracts. 

Methods

Isolation of the major catechins in green tea and the major curcuminoids in turmeric was completed via extractions. Green tea leaves were extracted, and the crude material was analyzed using UPLC-MS and analytical standards to identify the catechins of interest and develop a suitable prep-LC method for isolation. Turmeric powder was extracted and then analyzed by TLC-MS using Advion’s Plate Express™ to identify the compounds of interest and develop a suitable flash chromatography method for isolation. Both extracts were purified using mass-directed fraction collection using Interchim’s puriFlash 5.250 flash/prep chromatography system connected to Advion’s expression® single quadrupole mass spectrometer. Target compounds were detected using XIC MS channels. Purity of the isolated compounds was then determined using HPLC-MS.

Preliminary Data

We were able to successfully isolate the 3 major curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) in turmeric and the 5 major catechins in green tea ((-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-epigallocatechin-3-gallate (EGCG), (-)-epicatechin-3-gallate (ECG), and (-)-gallocatechin gallate (GCG) with high purity (≥95%).   

An isocratic flash chromatography method (97:3 DCM:MeOH) was developed using TLC (97:3 DCM:MeOH) to purify the curcuminoids. The TLC plate was analyzed by APCI– MS using the Plate Express which extracts spots directly from the plate with no need for sample preparation. Fractions were collected using extracted ion chromatogram (XIC) channels with APCI– MS. Fractions were then characterized by ASAP– MS and purity for each fraction was determined using HPLC-MS. 

A preparative LC method was developed for the catechins in green tea using HPLC-MS and reference standards to identify each compound of interest. Using a water methanol gradient and collecting fractions using XIC channels set for the compounds of interest. Fractions were characterized and their purity determined by HPLC-MS. EGC, EGCG, GCG, and ECG fractions were determined to have purities of 100 %, 99.8 %, 98.8, and 100 % respectively.

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Introducing the Advion Interchim Scientific SOLATION® ICP-MS

Introduction

The SOLATION® Inductively Coupled Plasma Mass Spectrometer (ICP-MS) puts the power of trace, multi-elemental analysis in your hands by simplifying and optimizing the typical ICP-MS workflow, inside and out. The system offers high performance, multi-elemental analysis, ideal for environmental, clinical, biomedical, food, agriculture, and geological applications.   

The SOLATION® offers a state-of-the-art quadrupole deflector that ensures the analyzer and detector stay clean and improves S/N by preventing neutrals and particles from entering the analyzer. The system is also designed for lower argon consumption. 

Key System Infrastructure Includes:

  • Ion extraction cones: Triple-cone ion extraction, followed by an Einzel lens, which are electrically controlled to maximize transmission of ions into the vacuum system. 
  • RF coil: Plasma generation with water cooled RF coil using industry standard 27 MHz variable frequency generator for rapid impedance matching and ultimate performance with challenging matrices. 
  • Torch: One-piece, demountable torch with fast, one-step connection of argon and ignitor. Optional shield to prevent secondary discharge. 
  • Nebulizer: High efficiency concentric nebulizer available in glass or quartz for compatibility with the widest range of flow rates and sample composition. 
  • Spray chamber: The cyclonic spray chamber with optional temperature control further reduces droplet size and solvent load to ensure stable, efficient plasma. 
  • Peristaltic pump: Integrated 4-channel, 12-roller pump for maximum flexibility and ultra-low pulsation. Software controlled flow rate from 1 μL/min to >1 mL/min. 
  • Gate valve: Allows quick and easy maintenance and replacement of the cones whilst maintaining vacuum integrity. 
  • 90° Quadrupole deflector: Ensures that the analyzer and detector are not in line with the plasma beam, preventing neutrals and particles from entering the analyzer, improving S/N and preventing contamination. 
  • Octupole collision cell: Acts as an ion guide and a collision cell with He gas to provide Kinetic Energy Discrimination (KED) to remove interferences. 
  • Quadrupole Analyzer: High frequency mass filter design with the highest stability to simultaneously maximize transmission, resolution, and abundance sensitivity. 
  • Dual function detectors: Measures in both analog and pulse detection modes with seamless transmission between the two, to allow measurement of high and low levels in a single analysis with more than 9 orders of magnitude of linear dynamic range. 
  • Pulse Detection: captures ions generating pulses shorter than 20 ns; accurate and linear to minimum dwell time of less than 100 μs 
  • Analog Detection: used for higher ion signals while pulse detection is deactivated to extend detector lifetime. 
  • Mass Dependent software control: Software designed to optimize specific mass ranges independently to allow for mass specific tune optimization.  

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Analysis of Heavy Metals in Cannabis using the SOLATION® ICP-MS

Instrumentation: SOLATION® ICP-MS

INTRODUCTION

With the growing acceptance and legalization of hemp and cannabis in the US, Canada, and several other countries, cannabis products are more widely available than ever before. Now approved for medical, recreational and health supplement uses, increased production and consumption have highlighted the need for routine testing and development of testing standards for toxic chemicals, including heavy metals, in cannabis plant material, and all the byproducts made from them, to ensure safe products for the consumer.  With the adoption of chapters <232> and <233>, US Pharmacopeia (USP) specifies a list of elements and maximum exposure limits based on toxicity and routes of administration for pharmaceutical products.  

Many states that have legalized medical and recreational cannabis base their exposure limits on the USP values.  California, Colorado, and Massachusetts are examples with Permissible Daily Exposure (PDE) limits by inhalation for As, Cd, Hg, and Pb. These values are summarized in Table 1. 

Table 1: PDE limits for states that use the USP guidelines for heavy metal exposure by inhalation. USP<233> also defines the accuracy, repeatability, and ruggedness required for the analysis of these toxic elements:

Validation Criteria

Accuracy: The matrix and materials under investigation must be spiked with target elements at concentrations that are 50%, 100%, and 150% of the maximum permitted daily exposure (PDE). Mean spike recoveries for each target element must be within 70%-150% of the actual.

Repeatability: Six independent samples of the material under investigation must be spiked at 100% of the target limits defined and analyzed. The measured percent relative standard deviation (%RSD) must not exceed 20% for each target element.

Ruggedness: Carrying out the repeatability measurement testing procedure by analyzing the six repeatability test solutions either on different days, either with a different instrument or by a different analyst. The %RSD of the 12 replicates must be less than 25% for each target element.

In this study, we used the Advion SOLATION® ICP-MS and a microwave digestion system to digest and analyze hemp samples using the validation methods described in USP General Chapter <233>.  The sensitivity, ability to handle complex matrices, and the ability to remove interferences with a helium collision cell makes this the ideal system for heavy metal analysis in the cannabis industry.

EXPERIMENT

Sample Preparation

A sample of medicinal hemp flower was purchased locally.  Around 14 grams was finely ground and homogenized, then 0.5g +/- 0.005g was weighed into digestion vessels and the appropriate amount of a spike solution added.  Nine mL of concentrated HNO3 and 1 mL concentrated HCl was added to each vessel and the samples were allowed to react for 15 minutes prior to sealing and placing the vessels on the turntable of the closed vessel microwave digestion system.  The program controls the microwave energy such that the samples ramped up to the optimal digestion temperature of 200°C over 20 minutes, maintained 200°C for 10 minutes, and then cooled back to room temperature.  

This method resulted in the complete digestion of all samples resulting in a clear, particulate-free solution once brought to volume with 18MΩ water in a 50mL volumetric flask.

The calibration standards and spikes were based on the action levels in table 1.  The sample set included a hemp sample, a duplicate, the 50%, 100%, and 150% spikes, and NIST 1575 Pine needles to further validate the results, which were run in duplicate.  For the ‘ruggedness’ specification from USP<233>, there were 6 samples of the 100% spiked hemp.  The spike values are summarized in Table 2.

Table 2: Spike values based on the action limits defined in USP<233>

A second, 1:4 dilution was performed post digestion to bring the final acid concentration to 5% for an overall dilution factor of 400x.  The calibration blank and standards were prepared using the same acid concentration, 5% of 9:1 HNO3/HCl, for matrix matching.  To stabilize mercury and help with wash-out, Gold was added at 20x the mercury concentration.  The internal standards were added to all the samples, standards and blank for a final concentration of 10 ng/g (ppb).  The standard concentrations and internal standards are summarized in Table 3.

Table 3: Analyte masses, calibration standards, and internal standards.

Instrumentation

The Advion SOLATION® ICP-MS incorporates a robust solid-state generator, orthogonal ion optics for keeping the most sensitive components of MS clean, and easy-to-use control and data processing software. 

Since HCl was used in the sample digestion, there is a significant amount of chlorine present which creates an isobaric interference on 75As from 40Ar35Cl+.  The collision cell effectively eliminates the contribution that ArCl+ makes to the signal at m/z 75 by taking advantage of kinetic energy discrimination (KED) to separate polyatomic interferences from analyte ions resulting in accurate quantitation of low levels of Arsenic.  Arsenic is the only analyte in the suite with this type of interference, so the collision cell isn’t used for Cd, Hg, or Pb.  

A glass concentric nebulizer fitted to a cyclonic spray chamber, connected to the standard torch with an injector ID of 2mm, was used for sample introduction.  Instrument running parameters are summarized in Table 4.

Table 4: ICP-MS Parameters

RESULTS AND VALIDATION

Sample Results

The concentrations of mercury and lead in hemp were less than the lowest standard and all values were less than the action limits. The samples were prepared and analyzed in duplicate, and the average of those duplicates is shown in Table 5. In keeping with the ruggedness requirement, the samples were run on separate days by two different analysts.

Table 5: Hemp sample results (average of the sample and duplicate)

Accuracy: The samples were spiked at 50%, 100%, and
150% of the action level (Table 2 above) and the percent recoveries calculated. Spike recoveries were all between 92.5% – 114.1%, well within the 70-150% range defined by the USP method.

Table 6: Accuracy- Spike recoveries

Repeatability: Six hemp samples were spiked at 100% of the action level and digested. The results that are summarized in Table 7 show that the %RSD of the measured concentrations are between 1.3% – 3.7%, demonstrating repeatability well below the 20% limit.

Table 7: USP<233> Repeatability results

Ruggedness: The repeatability sample set was prepared and run on a different day by a different analyst. The results from that run are combined with the previous run to determine the ruggedness. The ruggedness values are similar to the repeatability values and the measured %RSD (2.4 – 4.0%) are comfortably under the 25% limit defined by the USP method. The results are summarized in Table 8.

Table 8: USP<233> Ruggedness results

NIST 1575a Results

The results for the NIST SRM are summarized in Table 9. The values for As and Hg were less than
the low standard in solution but there is good agreement between the experimental values and the certified values.

Table 9: NIST 1575a Pine Needles SRM

CONCLUSION

This study demonstrates that the Advion SOLATION® ICP-MS , coupled with a microwave digestion system, is suitable for the accurate, robust and reproducible analysis of heavy metals in hemp plant material – greatly exceeding the requirements of USP <233> protocol.

Validation of the microwave digestion method was reinforced by the excellent recovery results obtained for the NIST SRM 1575 Pine.

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Direct Sample Analysis of Fizzy Drinks Without Sample Preparation on a Compact Mass Spectrometer

Mass Spec: expression® CMS
Sampling: ASAP

INTRODUCTION

Chemists are tasked with quickly identifying compounds created, ensuring quality of products, or evaluating safety. Current techniques are adequate, but not all offer the speed, data quality, or ease-of-use provided by the Advion expression® CMS. The CMS with the Advion ASAP offers chemists the ability to rapidly analyze solids, liquids and powders without tedious and time consuming sample preparation.

METHOD

The extended glass capillary of the ASAP was dipped into each fizzy drink sample. The excess was wiped off and the probe was inserted directly into the ASAP-enabled APCI ion source of the CMS, producing results in seconds.

Figure 1: The ASAP containing the sample directly inserted into the ASAP-enabled APCI source of the CMS for analysis.

Figure 2: Schematic of the ASAP sampling probe for APCI-CMS analysis.

RESULTS

SUMMARY

The ASAP/CMS analysis provided data in < 1 min with no sample preparation and no chromatography, making it ideal for reaction monitoring, compound identification, food safety, and analysis of natural products.