NESACS
ACS Fall 2023
Applied Pharmaceutical Chemistry Conference
Spring Research Festival
Blood Analysis using the Advion Interchim Scientific SOLATION® ICP-MS
Introduction
Trace elements are essential for proper biological functions in humans, and differing levels of trace elements are indicative of many diseases and conditions. Non-essential trace elements are also present in the human body as a result of environmental contaminants generated by human or industrial activities deposited in soil, air, water, and foodstuffs. These essential and non-essential trace elements are easily measured and monitored in blood, serum, and urine using ICP-MS.
In this application note we present a fast method for routine analysis of small volume blood samples for key toxic and essential elements using the SOLATION® Inductively Coupled Plasma Mass Spectrometer (ICP-MS) using a simple “dilute and shoot” sample preparation. The high sensitivity and wide dynamic range of ICP-MS are particularly important for the determination of trace levels of heavy metals, while simultaneously measuring nutritionally relevant elements at higher levels. Blood is a complex matrix, rich in proteins and salts that favor the formation of carbon- and chlorine-based interferences that affect many of the analytes. The collision cell on Advion Interchim Scientific’s SOLATION® ICP-MS is necessary for overcoming those interferences.
The SOLATION® ICP-MS has 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. 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.
Experiment & Results
Reagents & Materials
Nitric acid (Aristar Plus, trace metal grade)
Triton X-100 (especially purified, Roche chemical)
Water, type 1 (18.2 MΩ, Elga point of use system)
Methanol (hypergrade for LC-MS, Supelco)
Mg, Ca, Mn, Cu, As, Se, Cd, Pb, Ge, Rh, Au, and Ir standard solutions (1000 μg/ml, Claritas ppt grade)
Trace Elements Whole Blood L-1 (SRM1) (Seronorm)
Trace Elements Whole Blood L-2 (SRM2) (Seronorm)
Trace Elements Whole Blood L-3 (SRM3) (Seronorm)
Blank Whole Blood (WB(F)) (UTAK)
Acid diluent: (0.5% Nitric acid, 0.05% Triton X-100, 2% Methanol, 0.25 μg/mL (ppm) Au, and the internal standards: 10 μg/L (ppb) Ge, Rh, and Ir)
Table 1: Calibration and standard concentrations
Standards
The method was developed to determine Mg, Ca, Mn, Cu, As, Se, Cd, and Pb in whole blood samples. The elements were chosen to represent some of the commonly measured metals in blood, covering both essential and toxic elements. Standards were prepared using the acid diluent with elements at four concentration levels to cover the range typically seen in blood. Table 1 outlines the standard concentrations at each level and which elements are at that level. The analysis mode and the internal standard used for each analyte are in Table 2. The ICH guidelines call for a minimum of five concentrations to establish linearity; with the calibration blank we use six, satisfying this requirement.
Table 2: Analysis Mode and Internal Standards
Samples & Preparation
Samples were prepared in 15mL, metal-free centrifuge tubes. Acid diluent (14.7mL) was added to each tube, followed by 0.3mL blood sample for a 1:50 dilution. The tubes were capped and inverted 3-5 times to thoroughly mix.
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 running conditions for the instrument are summarized in Table 3.
Table 3: ICP-MS Operating Parameters
Results & Discussion
We followed the ICH “Validation of Analytical Procedures” guidelines for method validation which defines specific requirements for accuracy, precision (repeatability), detection limit and method detection limit (DL and MDL), and quantitation limits (LOQ).
The accuracy of the method was established using the Seronorm reference materials, which had certified concentration values for all the elements in the method. These materials were measured in triplicate and the analytical results were compared to the certified values and 95% confidence limits provided by Seronorm.
These values are plotted on Figure 1 where the minimum and maximum certified values are represented as a box. Our analytical values are plotted in Figure 1, and in every instance, our values lie inside the limits indicating a high degree of accuracy which easily meets the ICH specifications.
A second measure of accuracy is spike recovery. The “blank blood” (WB(F)) sample from UTAK was spiked with 150μL of the stock standard solution for all elements except Ca and Mg in the 15mL tube. The recovery is calculated as:
Spike recoveries are shown in Table 4. All recoveries are within 90 – 110%, indicating excellent recovery of the spiked analytes.
Table 4: Spike Recoveries
Method precision is measured as the repeatability of six Seronorm level 2 (SRM2) samples. Six samples of Seronorm level 2 (SRM2) were prepared, diluted, and analyzed individually. The results show less than 3% RSD among the replicates. The %RSD of the six replicates is presented in Figure 2.
Figure 1: Accuracy of Certified Seronorm Material Analysis
Figure 2: Precision of Seronorm Material Analysis
The method detection limit (MDL), and limit of quantitation (LOQ) were determined using the standard deviation (σ) of the signal from eight acid diluent blanks. The acid diluent blanks were prepared using the same technique and equipment as the samples and were included at the end of each run followed by a calibration standard. The MDL and LOQ are calculated as:
where S is the calibration slop, and:
Since the calibration slope was the analyte relative to an internal standard, the calibration standard was used to determine the counts/second/ppb. These values are calculated, multiplied by 50 to account for the dilution factor, and expressed in μg/L (ppb). In the table, the MDL and LOQ are plotted relative to physiological normal values represented by Seronorm 2 (SRM2). For most analytes, the MDL and LOQ are miniscule by comparison, particularly for the major elements calcium and magnesium. However, even for more challenging analytes such as selenium, the MDL is an order of magnitude less than Seronorm 2 (SRM2) which, despite being level 2 (SRM2), has the lowest selenium content of these three SRMs.
Figure 3: LOQ and MDL as compared to physiological normal values
*as represented by Seronorm L2, formulated to represent typical, or average human values.
Conclusion
In this application note, we report on the analysis of trace elements in blood using the Advion Interchim Scientific SOLATION® ICP-MS. Blood is a complex and challenging matrix. However, these data support the use of a straightforward “dilute and shoot” sample preparation method that gives accurate and precise concentrations for high level and trace level elements in blood. 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 blood for rapid, accurate and reproducible results.
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.
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.
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.