Minerals Flashcards
What are forms of Coppers and how are they absorbed?
Cu+ or Cu2+ , mainly Cu2+.
90% in plasma bound to caeruloplasmin
Absorption ↓ by Zn, which induces metallothionein, which binds Cu in intestine
What are functions of Copper?
- Cytochrome oxidase: mitochondrial electron transport chain)
- Superoxide dismutase: free radical scavenger, prevents against oxidative damage
- Dopamine hydroxylase: catecholamine synthesis
- Monoamine oxidase: catecholamine breakdown)
- Tyrosinase: melanin synthesis
What causes Copper Deficiency?
Caused by:
- Loss from body in bile
- Chronic malabsorption resulting in diarrhoea;
- With jejunostomies
- Rarely genetic disorders of copper transport (Menke’s disease: ATP7A gene)
What are Symptoms of Copper Deficiency?
- Anaemia
- Neutropenia
- Bone changes similar to osteoporosis
- Hair changes,
- Neurological (neuropathy, myelopathy)
What causes excess in Copper and their signs/symptoms?
Caused by Contaminated drinking water (copper pipes) and Wilson’s disease (ATP7B gene)
Symptoms/Signs:
- Kaiseer Fleischer rings
- Cirrhosis
- Parkisonian disease
What are features of Zinc?
- Highly bound in serum to α2 macroglobulin and albumin
- Zinc is required for the activity of many enzymes, including those in DNA and protein synthesis.
- Zinc is beneficial for wound healing in zinc-deficient subjects
- Body does not store Zn to any appreciable extent in any organ therefore need regular dietary supply
- Primary route of elimination is biliary; normally only small amounts is excreted in urine
What are causes of Zinc Deficiency?
- Catabolic states (after trauma, surgery, burns) lose Zn in their urine
- Chronic malabsorption syndromes
- Pregnancy
- Lactation
- Old Age
- Alcoholism
- Diuretics
- Chelating agents (eg. penicillamine for Wilson’s),
- Cancer
- Chemotherapy
- Acrodermatitis enteropathica (inherited defect in intestinal Zn absorption)
What are Symptoms of Zinc deficiency?
- Dermatitis
- Delayed wound healing
- Alopecia
- Diarrhoea
- Weight loss
What are features of Selenium?
- Constituent of Selenoproteins: Glutathione peroxidase (protects against oxidative damage) and Deiodinases (T4ÞT3)
- Selenium bioavailability higher in plant foods (absorbable forms Se-methionine, Se-cysteine) than animal foods
- Inorganic forms (selenite, selenate) in drinking water - less absorbable
What causes Selenium deficiency and what are their signs/symptoms?
- May be caused by malabsorption e.g. Crohn‘s and specialist diets e.g. for PKU.
Symptoms
- Leads to muscle weakness, cardiomyopathy
- In Keshan in China: low Se in soil associated with deficiency and susceptibility to cardiomyopathy
What are features of Iodine?
- Forms part of thyroid hormones: Thyroxine (T4) and Triiodothyronine (T3)
- Deficiency is an Important worldwide health problem resulting in mental retardation
- Number of mechanisms prevent toxicity, but persistently high intakes can cause hyperthyroidism
Who are tested for Vitamin and Trace element analysis?
- Patients on nutritional supplementation
- Patients who are malnourished
- Patients where there is clinical suspicion of overdose or deficiency
- Malabsorption syndromes e.g. Crohn’s disease
- Fat malabsorption syndromes e.g. pancreatitis, Zollinger-Ellison
- Congenital disorders e.g. Abetalipoproteinaemia
- Patients with increased loss e.g. haemodialysis
What is the causes of Acute Phase?
- Metabolic changes occur in the body in response to infection, trauma, tissue damage.
- Can result in changes in micronutrients which are unrelated to nutritional status
Caused by:
Redistribution of binding proteins
Increased uptake by tissues
What happens to Vitamin and Trace element analysis in Acute Phase Response?
- CRP >10mg/L – Plasma Copper, Selenium and Vitamin C are unreliable
- CRP >20 mg/L – Plasma Zinc, Vitamin A and Vitamin D are unreliable
How can Vitamins and Trace Elements be measured indirectly?
- Haemoglobin used as a surrogate of Iron status
- Iodine infrequently measured but thyroid hormone status often checked
- Cobalt may be measured as an indicator of vitamin B12 status
- Homocysteine can indicate vitamin B12/folate status. Homocysteine may be elevated when folate/B12 is deficient
- Methylmalonic acid (MMA) can indicate vitamin B12 status. Increased MMA may indicate B12 deficiency
- Prothrombin time (INR) indicates vitamin K status
What has to be done with sample taken for Vitamin and Trace element analysis?
Mainly serum/plasma samples which may have specific collection requirements
- Vitamins A, E, B2 and B6 are light sensitive
- Vitamin C is extremely unstable
Ideally fasting sample. If this is not possible, sample should be taken at least 8 hours post treatment for patients receiving parenteral nutrition
What are methods and features of Trace Element Analysis?
- Matrix: mainly Serum/Plasma
- Method of analysis: mainly HPLC or immunoassay
- Frequency of measurement: repeat 2-4 weeks after initial supplementation, but many vitamins do not require repeat testing for several months
- Not a role for routine measurement of some vitamins e.g. vitamin C
What are methods and features of Trace Element Analysis?
- Generally measured by ICP-MS
- Careful collection of samples is required
- Other analytes may be useful, e.g. caeruloplasmin is measured in conjunction with copper in the investigation of Wilson’s disease. Gives an indication of free copper available
What is the sequence for Inductively Coupled Plasma Mass Spectrometry?
- Ionisation source that fully decomposes a sample into its constituent elements
- Elements transformed into ions
- Usually made from argon gas
- Energy is coupled to it using an induction coil to form the plasma
- Ions are produced, sampled, filtered by mass and detected
What are features of Inductively Coupled Plasma Mass Spectrometry?
Highly sensitive analysis
Simultaneous analysis of metals at trace concentrations (pmol/L)
- Copper, Selenium, Zinc
- Toxic metals e.g. Lead
What is Plasma in the ICP?
- Plasma is a gas mixture at a high temperature containing ions, electrons and neutral particles. The charged particles allow energy transfer into the plasma via induction
- Self-sustaining with temperature >5000°C
How is Plasma formed?
- ICP is generated at the end of a quartz torch. Torch made of 3 concentric quartz tubes. Argon gas directed through torch
- Copper coil surrounds end of torch
- Connected to RF generator giving an oscillating magnetic field
- High voltage spark creates ion which move in the field and collide with other Ar atoms
- Collisions produce more ions which then move with the field and generate heat. Plasma is maintained as long as argon is added and rf current through loading coil is maintained.
How is the sample introduced?
Samples most commonly analysed as solutions introduced via a peristaltic pump via a nebuliser which produces a fine aerosol
Only smaller droplets pass into the plasma
Within the plasma droplets will undergo 4 processes:
- Solvent evaporation
- Vaporisation
- Atomisation
- Ionisation (by electron impact)
What is the sample interface?
- Plasma temperature is ~5000°C
- Adjacent ion focussing device operates at room temperature
- Also large pressure difference
- Sampling interface allows ions to travel into the lens region
- Traditionally consists of 2 water cooled metal cones separated by a region held under vacuum. Cones usually made of nickel.
- Sampler cone (~1 mm diameter): Samples central channel of plasma
- Skimmer cone (~0.7 mm diameter): Skims the beam to reduce gas load passing through to ion focussing region and MS
- Ions, photons and neutral atoms are extracted from the plasma into the interface region which has a pressure of 150 – 300 kPa
- The dramatic reduction in pressure causes a supersonic expansion of the ions
- Extracted by skimmer cone into main vacuum
- At this pressure ions can be guided to charged surfaces called electrostatic lenses
What are Ion Optics?
- Electrostatic lenses behind the skimmer cone are called the ion optics
- They Guide beam to mass analyser
- Prevents photons from reaching mass analyser as they are a source of signal noise
- Design differs between manufacturers
What is the Mass Spectrometer?
- Positive ions sampled from the plasma focused into the MS using a series of electrostatic lenses held at specific voltages
- Positive ions are separated according to their mass to charge ratio (m/z)
- Ion optics and mass spectrometer both under high vacuum
- Three basic mass spectrometer types are available for ICP-MS: Quadrupole, Magnetic sector field (high resolution), Time-of-flight (TOF)
- Most common type is the Quadropole
What is a Quadropole?
- 4 cylindrical rods diagonally paired. A quadrupole is essentially a mass filter separating ions on their m/z ratio
- Voltages of opposite charge applied to each pair of rods which creates an oscillating field within the quadrupole
- A series of lenses direct ion beam along the central axis of the quadrupole
- At any given set of voltages, only ions of a certain m:z ratio will have stable trajectories along the axis of the quadrupole, and reach the detector
How is detection undertaken in the ICP-MS?
- Ions can’t be detected directly - their signal must first be amplified
- Most instruments use ‘discrete dynode’ ion multipliers to achieve this
- A channel electron multiplier set at negative voltage provides the attractive force for the positive ions
- Ion strikes the surface of the detector (first dynode) and its impact causes the release of electrons. These are accelerated into a second and subsequent dynodes, releasing more electrons (avalanche effect)
- End result is an electron cascade (pulse) at the multiplier exit, for each incident ion
What are the steps for Data Acquisition?
Scanning
- All measurable masses acquired
- Qualitative and quantitative analysis
- Can retrieve data post-run for un-calibrated elements
Peak jumping
- Selected isotopes only
- Optimised dwell times (gives improved detection limits)
Time resolved analysis
- ICP-MS interfaced with HPLC
- Transient signal analysis
- Applicable to laser ablation ICP-MS and chromatographic work (speciation)
What is Abundance Sensitivity?
- Contribution that a signal for an isotope at a certain mass (M) makes to adjacent masses (i.e. M-1 and M+1). It is essentially a measure of peak tailing
- Mass peaks usually have a slight negative skew
- Can result in interference when the signal at one mass is considerably higher than the signal at an adjacent mass
What is the internal Standard?
- Corrects for sample specific matrix effects
- Mass and ionisation potential of an element are two of the most significant determinants of matrix effects
- Ideal internal standard will have a similar mass and ionisation potential to the analyte (e.g. copper has an atomic mass of 63 amu and an ionisation potential of 7.73 eV Germanium has both a similar mass (72 amu) and ionisation potential (7.90 eV))
- Internal Standard should not suffer from spectroscopic interference from the sample matrix and should not itself cause spectroscopic interference with the analyte
What are common internal standards?
- Lithium
- Scandium
- Geranium,
- Indium
How are samples prepared for ICP-MS?
- Sample dilution and addition of surfactants e.g. Triton X-100 to reduce sample viscosity and prevent blocking of the torch injector tube
- Matrix matched standards
What is Semi Quantitative Calibration?
- Used when complete precision not required and analyte standards not available
- Concentration derived by calculating the ratio of the analyte response to a calculated sensitivity factor
- A reference element used to “calibrate” the measured signal for another element to provide an estimate of the concentration of the second element
- Result is corrected for abundance of the isotope measured and any variation in ionisation efficiency of the element
What is Fully Quantitative Calibration?
- Multi element external calibration standards
- Does not account for matrix induced effects (signal suppression or instrument drift)
- Standard additions calibration
- Isotope ratio determination
What are the 2 types of Interferences seen in ICP-MS?
- Spectroscopic Interferences: Non-analyte ions have the same m/z ratio as the analyte
- Non-spectroscopic interference: Non-spectroscopic interferences may be broadly divided into matrix effects and instrument drift
What are the 4 types of Spectroscopic Interferences?
- Isobaric elements
- Double charged ions
- Polyatomic ions
- Tailing interference
What are Isobaric elements?
Where 2 isotopes of different elements have the same mass (e.g.58Fe+ and 58Ni+, 114Sn+ and 114Cd+)
What are Double Charge Ion interferences?
- Most elements form singly charged ions in the ICP, however elements with a second ionisation potential lower than the first ionisation potential of argon also form a small but significant fraction of double charged ions e.g. Gadolinium
- Gadolinium used as contrast in MRI and can interfere with selenium measurement
- Rare interference
Describe Polyatomic Ions interference
- Arise from the formation of polyatomic ions in high temperature plasma, either due to incomplete atomisation or recombination during extraction of ions into MS
- Ions may be derived from the sample matrix, reagents used for sample preparation, plasma gases (argon) or entrained atmospheric gases E.g. in samples containing chloride (or where hydrochloric acid is used during sample preparation), chlorine oxide (35Cl16O) and argon chloride (40Ar35Cl) are formed in the plasma. These ions share the same m/z ratio as vanadium (51V) and arsenic (75As) respectively.
Describe Tailing Interferences?
- Spectral overlap from an adjacent mass
- Depends on the abundance sensitivity of the mass analyser.
- E.g. 55Mn which is bordered on either side by 54Fe and 56Fe
What are the types of Non-spectroscopic interference?
- Matrix effects
- Instrument drift
- Dilution
What are Matrix Effect interferences?
- Enhancement or suppression of analyte signal due to sample matrix due to several mechanisms
- Sample introduction e.g. nebulisation rate affected by viscosity and surface tension of the sample
- Plasma effects
- Space-charge effects
What are Instrument Drift interferences?
Gradual deposit of dissolved solids in samples in the nebuliser and interface cones
What are some dilution interferences?
Matrix effects are dependent on the absolute concentration of matrix components, therefore sample dilution (either during sample preparation or performed online) will reduce the severity of matrix effects
How can you undetake Spectral interference corrections?
- In some cases, can avoid interference by measuring another isotope (e.g. 82Se+ in place of 80Se+ to avoid 40Ar2+ interference)
- Can change sample preparation e.g. use an alternative to HCl
- Can be coupled to chromatographic separation
- Can mathematically correct for the interference using known isotopic abundances
- Physical correction (separation of interfering element in matrix from the analyte)
- e.g. gel filtration to remove NaCl and eliminate 23Na+40Ar+ interference with 63Cu+
- Chemical correction e.g. addition of propan-1-ol to decrease polyatomic species formation (used for selenium analysis in biological samples)
- Collision cells
What are collision cells?
- A means to remove spectral interferences in ICP-MS
- Positioned immediately before the quadrupole. A Collision Cell consists of quadrupole which is enclosed in a cell that can be filled with a gas e.g. Helium under positive pressure, and is located after the main ion lenses
- The “cell” gas reacts with interfering molecular ions either by charge transfer reactions, or by forming derivatives
- Results in dramatic reduction in abundance of interfering ions either by a chemical reaction process or reducing the kinetic energy of polyatomic ions
What are the collision modes in the collision cells?
- Works on the principal that interfering ions e.g. ArO+ are physically larger than the analyte ions
- If both ions are allowed to pass through a cloud of inert gas molecules, the interferent ion will collide more frequently with the inert gas atoms than will the analyte ion due to its larger size
- Each collision removes kinetic energy from the ion
- The analyte ion will therefore retain more energy than the interferent ions after passing through the collision cell
- Energy barrier at the exit of the collision cell can be adjusted so that higher-energy analytes are allowed to pass through to the detector
What are the reaction modes in the collision cells?
- Interferent ions react with active gas e.g. ammonia exothermally
- Analyte ions react with the same gas endothermally
- Interferent ions are converted to neutral atoms
- Neutral atoms do not carry a charge and are therefore not stable in the reaction cell and are rapidly ejected
- Analyte ions are not affected and pass through to the quadrupole
What are features of the Standard Mode in Collsion cells?
Detection Limits
Low
How it Works
Cell gas is turned off so there
is no loss in analyte sensitivity.
Actively vented cell design
ensures no residual gases remain, eliminating potential interferences and allowing quick switching to and from other modes.
Applications
Routine applications requiring
high throughput that have few
interferences.
Interference Removal
Technique
Correction Equations
Cell Gas(es)
None
What are features of the Collision Mode in collision cells?
Collision mode
Detection Limits
Lower
How it Works
Non-reactive gas is introduced into the cell to collide with the interfering ions with larger
diameters, reducing their kinetic energy so they may be removed through Kinetic Energy Discrimination.
Applications
Applications that may be
susceptible to interferences,
or analyses where you simply
want to remove any unknown
interferences.
Interference Removal
Technique
Kinetic Energy Discrimination
Cell Gas(es)
Non-reactive e.g. Helium
What are features of the Reaction Mode of Collision Cells?
Reaction mode
Detection Limits
Lowest
How it Works
Highly reactive gas (or gasses) is introduced into the cell to create predictable chemical reactions. Any side reactions and resulting new interferences are instantly removed by a scanning quadrupole.
Applications
Applications demanding the
very best performance and
an unprecedented level of
interference removal
Interference Removal
Technique
Dynamic Reaction Cell/ Scanning Quadrupole
Cell Gas(es)
Pure reactive e.g. Ammonia
