Group 8 mass spec automated analyser and poct the complete version

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MASS SPECTROMETRYAUTOMATED ANALYZER

POCTGROUP 8

By:Delgado, Sharmaine Kay

Gloria, Sherina AnnLagos, Riza Jane

Pillora, Gin AnilouVillaflor, Mary Queen

MASS SPECTROMETER

Contents:

• Mass Spectrometry• Mass Spectrometer• Principles• Major Parts• How it works• Uses• Types of Spectrometer

What is Mass Spectrometry?

• An analytical technique that measures the mass-to-charge (m/z) ratio of charged particles.

• A technique of separating and identifying molecules based on its mass.

What is a Mass Spectrometer?

• A mass spectrometer is an analytical tool used to determine the elemental composition of an unknown substance. It utilizes the charged particles of molecules to separate them.

Principles

Principles• A Mass Spectrometer

produces ions from the substance under investigation, separates them according to their mass-to-charged ratio (m/z) and records the relative abundance of each present.

Principles• Different elements can be

uniquely identified by their mass.

Principles

• Different compounds can also be uniquely identified by their mass.

Butorphanol L-dopa Ethanol

NOH

HO

-CH2-

-CH2CH-NH2

COOH

HO

HO

CH3CH2OH

MW = 327.1 MW = 197.2 MW = 46.1

Remember• The heavier the ion, the

lesser the deflection.

• The lighter the ion, the greater the deflection.

Major Parts and Function

• Mass spectrometers consist of four basic parts;

• a handling system to introduce the unknown sample into the equipment;

• an ion source, in which a beam of particles characteristic of the sample is produced;

• an analyzer that separates the particles according to mass; and

• a detector, in which the separated ion components are collected and characterized.

http://en.wikipedia.org/wiki/File:Mass_spectrometer_schematics.png

Major Parts of a Mass Spectrometer

1.Inlet The sample to be

analyzed enters the instrument through the inlet, usually as a gas, although a solid can be analyzed if it is sufficiently volatile to give off at least some gaseous molecules.


Major Parts of a Spectrometer

2. The Ionization ChamberIn the ionization chamber, the

sample is ionized and fragmented. This can be accomplished in many ways—electron bombardment, chemical ionization, laser ionization, electric field ionization—and the choice is usually based on how much the analyst wants the molecule to fragment.



3. The Mass Analyser Here, the particles are separated

into groups by mass, and then the detector measures the mass-to-charge ratio for each group of fragments by electromagnetic fields.



4. The DetectorFinally, a readout device—

usually a computer—records the data.


How it Works

How it Works


Stage 1:Ionisation Source

• The Sample is vaporized into gas for ionization,

• The atom is ionised by knocking one or more electrons off to give a positive ion.

• The Ion source is maintained in a high vacuum environment to enhance collision efficiency and ion formation.


Stage 2:Acceleration

The ions are accelerated so that they all have the same kinetic energy.


Stage 3:Deflection

• The ions are then deflected by a magnetic field according to their masses. The lighter they are, the more they are deflected.

• The amount of deflection also depends on the number of positive charges on the ion - in other words, on how many electrons were knocked off in the first stage. The more the ion is charged, the more it gets deflected.


Stage4:Detection

The beam of ions passing through the machine is detected electrically.


Types of Mass Spectrometer

1. GC/MS (Gas Chromatography-Mass Spectrometry)

• Is a method that combines the features of Gas-Liquid Chromatography and Mass Spectrometry to identify the different substances within a sample.

2. AMS (Accelerator Mass Spectrometry)

• a ‘’tandem accelerator’’ is used to accelerate the ions at several million volts.

3. ICP-MS (Inductively Coupled Plasma-Mass Spectrometry)

• involves the formation of gas containing electrons, ions and neutral particles from Argon gas. The sample is atomized and ionized by this gas. In a high vacuum mass analyzer, these ionized atoms from gas are passed through cones (apertures).

4. IRMS (Isotope Ratio Mass Spectrometry)

• It is used to measure mixture of stable isotopes. It has two inlets that help in repetitive measurements with continuous supply of sample gas.

5. Tandem MS (Tandem Mass Spectrometer)

• is a spectrometer used to separate ions based on a sample’s ‘’electronic’’ mass using two or more quadruple’s

6. TIMS (Thermal Ionization-Mass Spectrometry)

• is a mass spectrometer that can make exact measurements isotope ratios of thermally ionisable elements. This ionization can be done by passing them through metal ribbons under vacuum.

7. SSMS (Spark Source Mass Spectrometry)

• can ionize the analytes in solid samples using electric current with two electrodes. It works as one electrode if the sample is metal or can be placed in a cup-shaped electrode by mixing with graph detected isotopes from the sample.

8. (LC/MS or LC-MS) Liquid chromatography –mass

spectrometry • It is used to separate

compounds chromatographically before they are introduced to the ion source and mass spectrometer. LC-MS is a powerful technique used for many applications which has a very high sensitivity and selectivity.

9. IMS/MS or IMMS (Ion mobility Spectrometry)

• Is a technique where ions are first separated by drift time through some neutral gas under an applied electrical potential gradient being introduced into mass spectrometer.

Interpretation of Results

Advantages

• Fast• Differentiates Isotopes• Can be combined with GC and LC to run mixtures


Disadvantages

• Doesn’t directly gives structural information.• Need pure compounds• Difficult with non-volatile

compounds


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Automated Analyzer

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Principles of Automated Analyzer

Automated analyzers process large volume of tests with great precision and speed.

It permits the operator to focus on tasks that cannot be readily automated and increased both efficiency and capacity.


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Steps in Automated analysis:

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Basic Types of Automated Analyzers

Continuous Flow Analyzers

Discrete Analyzers

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I. Continuous Flow Analyzer

Pumped through a system of continuous tubing. Samples are introduced in a sequential manner, following each other through the same network.

This analyzer is capable of analyzing one analyte at a time.

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Principle of Continuous Flow

Analyzer

An essential principle of the system is the introduction of air bubbles.

Function of Air Bubbles: The air bubbles segment each sample into

discrete packets and act as a barrier between packets to prevent cross contamination as they travel down the length of the tubing.

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The air bubbles also assist mixing by creating turbulent flow and provide operators with a quick and easy check of the flow characteristics of the liquid.

Function of Air Bubbles:

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Continuous Flow Analyzer Instrumentation

In Continuous Flow Analysis a continuous stream of material is divided by air bubbles into discrete segments in which chemical reactions occur.

The continuous stream of liquid samples and reagents are combined and transported in tubing and mixing coils.

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Continuous Flow Analyzer Instrumentation

The tubing passes the samples from one apparatus to the other with each apparatus performing different functions, such as distillation, dialysis, extraction, ion exchange, heating, incubation, and subsequent recording of a signal.

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Continuous Flow

Continuous flow is used in some spectrophotometric instruments in which the chemical reaction occurs in one reaction channel and then is rinsed out and reused for the next sample, which may be an entirely different chemical reaction.

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Continuous Flow

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Types of Continuous Flow Analyzer

Segmented Stream System-The reaction stream is segmented

with bubbles of air or nitrogen to reduce inter-sample dispersion.

Flow Injection Analysis- It is low pressure and without

separation. The injected sample mixes and reacts with the flowing stream.

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Instrumentation of Segmented

Stream SystemIt includes a peristaltic pump that continuously aspirates sample and reagent, a variable number of tubes constituting a manifold to circulate liquid and a detector system.

Aspirated sample are segmented by injecting air bubbles that should be remove before they can reach to the detector.

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Instrumentation of SegmentedStream System

At detector air bubbles are removed and each sample is separated by washing solution, thus a square shaped detector response is obtained, the height of rectangle is directly proportional to concentration of analyte.

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Segmented Stream System

Reaction coil

Separation unit

Debubber

Air Sampler

Pump

Diluent

Reagent

Waste

Air

Detector

Flow-cell

Thermostatedbath

Reaction coil

Reaction coil

Separation unit

Debubber

Air Sampler

Pump

Diluent

Reagent

Waste

Air

Detector

Flow-cell

Detector

Flow-cell

Thermostatedbath

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Flow Injection Analyzer

FIA is based on the injection of a liquid sample into a moving continuous non segmented carrier stream of a suitable liquid. The injected sample forms a zone which is then transported towards a detector.

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Mixing with reagent in the flowing stream mainly occurs by diffusion-controlled process and a chemical reaction occurs.

Detectors continuously record the physical parameter as it changes as a result of passage of sample material through flow cell.

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II. Discrete Analyzer

Discrete analysis is the separation of each sample and accompanying reagents in a separate container.

Discrete analyzers have the capability of running multiple tests on one sample at a time or multiple samples one test at a time.

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Discrete Analyzer

They are the most popular and versatile analyzers and have almost completely replaced continuous-flow and centrifugal analyzers.

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Instrumentation of Discrete Analyzer

Sample reactions are kept discrete through the use of separate reaction cuvettes, cells, slides, or wells that are disposed of following chemical analysis.

This keeps sample and reaction carryover to a minimum but increases the cost per test due to disposable products

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Samples are applied to slides that are automatically dispensed from test- specific cartridges. Sample application is performed by means of individual, disposable tips, thereby eliminating the carryover problem. The sample itself provides the liquid necessary to hydrate the reagent layers of the slide.

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The slides incubate in heated air chambers and the color that develops is measured by reflectance photometry from the bottom side of the slide.

Results for each sample are collated and printed in a report form that could be suitable for use as the final chartable report.

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Discrete Analyzer

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Batch Testing- Samples are processed in concert as a group or “batch” in the same analytical analysis.

Sequential Testing – samples are processed sequentially rather than in a batch.

Designs of Analyzer Pathway

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Parallel Testing- samples undergo a series of analytical processes, usually for one analysis at a time, often used with batch analysis.

Random access testing- a system where any specimen can be analyze in any sequence with regard to the initial order of the specimens.

Designs of Analyzer Pathway

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Major Components of Automated Analyzers

Patient Identification Sampling Sample and Specimen

Transport Dilution Mixing Incubation Reaction Vessels Analysis of Measurement Data Analysis

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Patient Identification

Patient identification was accomplished by transcribing patient information onto sample cups and print outs of test results.

With the arrival of computers, the operator could input patient information to the laboratory computer.

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Bar code labeling systems are now employed. The bar code was read and would match patient data with test results. The use of bar code labels has served to reduce errors in matching test results with the proper patient.

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Sampling

Accomplished by syringe pipette or aspirating probe. The specimens are transferred to sample cup, and the sample pickup device aspirates the specimen.

In CFA, the aspirating probe is dipped into the sample cup and the specimen is drawn up using a peristaltic pump.

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Sampling-Dispensing Automatic Pipets

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Peristaltic Pump

A peristaltic pump is a type of positive displacement pump used for pumping a variety of fluids.

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Peristaltic Pump Principle

Works by squeezing the tube with rollers/shoes. It can run dry, self-prime and handle viscous or abrasive liquids, plus, as the tube is one complete unit, there are no seals thus making the pump leak free and hygienic. Excellent for dosing applications. Although this principle applies to all peristaltic pumps the difference is in the head and the drives.

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How Does a Peristaltic Pump Work

As the rollers and wiper move, a part of the tube is pressed, causing the fluid to be pumped onward. A restitution fluid can be sent into the pump as the rotors and rollers moved back the process is called 'Peristalsis‘. It forms the basic function within a Peristaltic Pump.

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Peristaltic Pump

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Piston Pump

A piston pump (reciprocating pumps) is a type of positive displacement pump where the high-pressure seal reciprocates with the piston. Piston pumps can be used to move liquids or compress gases. Powered by an electric motor, steam or a turbine, hydraulic drive mechanism.

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Function

A piston pump uses the reciprocating motion of a piston rod to move fluid along an axis through a cylinder-shaped chamber. As the piston moves through the cylinder, pressure builds up and forces the fluid through the pump. The fluid flowing through the pump pulsates due to the movement of the piston through the cylinder.

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Piston Pump Operating Principle

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Advantages of Piston Pumps

• Reciprocating pumps will deliver fluid at high pressure (High Delivery Head).

• They are 'Self-priming' - No need to fill the cylinders before starting.

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Sampling

Discrete analyzers employ a variety of syringe pipettes to aspirate and dispense sample and reagents. An important consideration for any sampling device is specimen carry-over and therefore it should be designed to reduce this problem.

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Sample and Specimen Transport in CFA

In continuous flow analyzers, specimen transport is accomplished using the peristaltic pump. Air bubbles separate aliquots of the same sample and isolate one specimen from another.

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Sample and Specimen Transport in Discrete

Analyzers In the Dupont aca, the sample reagent

pack is transported throughout the analyzer with a chain-driven pulley system.

Some analyzers used a motorized carousel, for example, the Olympus Demand, to move the reaction vessel in a circular path within the instrument.

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Sample and Specimen Transport

The Kodak Ektachem analyzers meters the sample aliquot, by use of a disposable sample tip secured by an apparatus called proboscis, onto a slide for transport to incubation chambers and detectors.

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Dilution

Sample and reagent dilutions are usually accomplished with the syringe pipettes and pumps. The pumps must be designed to aspirate and deliver precise volumes of fluid.

The dilution volumes maybe adjusted by use of a cam or programmed via a microprocessor as seen in many discrete analyzers.

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Mixing

In an automated system such as continuous analyzer mixing of a sample and reagents is accomplished using a glass coil inserted into the flow path. As the sample mixture passes through the coil, it is inverted and mixed via gravity.

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Mixing in Discrete

Analyzers In the Beckam ASTRA systems, a

magnetically driven Teflon stirring bar located in the bottom of the reaction chamber is used.

The DuPont aca employs a breaker mixer that mechanically vibrates and shakes the pack.

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Incubation

Reaction mixtures that require incubation must be conducted at constant temperatures without significant fluctuations. a.) heating the air around the cuvetteb.) heating metal blocksc.) using water baths.

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Reaction Vessels

In CFA systems the tubing serves as reaction vessel.

In DA, any of the following maybe used:a.) The DuPont aca uses a sealed plastic

bag that also serves as the cuvette. b.) The Teflon or plastic rotors in

centrifugal analyzers serves as the reaction vessels.

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Reaction Vessels

c.) Hitachi series and Baxters Paramax 720 ZX use plastic cuvettes. d.) Eastman Kodak Ektachem uses a multilayer thin film slide. Each slide is impregnated with reagents. Sample cup via a disposable pipette tip onto the slide that also serves as the cuvette for the reflectance or electrochemical measurement.

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Data Analysis Light-emitting diodes offer direct readout

of absorbance and replace the earlier recorders with an ink pen to trace the response of the phototube on paper.

Computer in the laboratory instrumentation allowed users to display results in a variety of formats and printers provide a hard copy of patient’s results.

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Data Analysis

Calculations, calibration curves, and quality control are performed by the computers, thus reducing errors and providing more accurate results than a non-computerized instrument.

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Analysis of Measurement Most automated chemistry analyzers

use photometric methods of analysis such as spectrophotometry, fluorometry, nephelometry, and reflectometry.

Some analytes, for example sodium and potassium, require the use of electrochemistry for analysis.

Instrument manufacturer have designed electrochemical devices based on coulometry, amperometry, and potentiometry to measure these and other analytes.

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Analysis of Measurement

Automated systems based on colorimetry use narrow-band interference filters for the isolation of specific wavelengths. The filters are contained in a circular disk, called a filter wheel, that rotates into the light path. A computer controls the rotation of the filter wheel and multiple wavelengths can be use to analyze a specimen.

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Automated Analyzers determine the levels of:

• Albumin• Alkaline

phosphatase• Aspartate

transaminase (AST)• Blood urea

nitrogen, • Bilirubin

• Cholesterol• Creatinine• Glucose• Inorganic

phosphorus• Protein• Uric acid in bloods• Calcium

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Advantages

Increase the number tests performed by one medical technologist in a given period.

Minimize the variation in results from one medical technologist to another.

Automation eliminates the potential errors of manual analyses as a volumetric pipetting steps, calculation of results, and transcription of results.

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Advantages

Instruments can use very small amounts of samples and reagents.

Reduction in the variability of results and errors of analysis through the elimination of task that are repetitive and monotonous for most individuals.

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Advantages Faster analyses up to 120 samples per hour Automatic data recording and preparation Being a closed system, automation reduces

contamination Greater accuracy and reproducibility of

results as all samples are subject to same processes

Smaller sample and reagent volumes, reduces cost

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DISADVANTAGES Time-consuming sample preparation

steps such as distillations, digestions, and matrix removal or enhancement performed manually before testing by a discrete analyzer.

Cannot perform complex chemistries such as on-line gas diffusion, dialysis, distillations, extractions, and digestions

POINT OF CARE ANALYZER

Point of Care Testing• is defined as medical

testing at or near the site of patient care outside of the conventional laboratory. .

• brings the test conveniently and immediately to the patient and increases the possibilities of the patient receiving the test result in a timely manner.

PRINCIPLE:

• point-of-care test systems are easy-to-use membrane-based test strips, often enclosed by a plastic test cassette.

• These tests require only a single drop of whole blood, urine or saliva, and they can be performed and interpreted by any general physician within minutes.

• • POCT are accomplished through the use of

transportable, portable, and handheld instruments and test kits.

POCT METHODS:

• Non-automated Methods- may be done by manual rapid-testing methods using a Dipsticks or Immunostrips.

• Instrument-Based and Automated Methods- are automated and use a small amount of specimen. This type of automation requires minimal technical support and is easy to use. It includes visual readings, display screen, printer, infrared, wireless radio signals, or modems.

• Most of the instruments utilized for POCT use whole blood for analysis and disposable reagent unit-dose devices.

• The most popular POCT instrument is the I-STAT analyzer.

I-STAT ANALYZER

It is a portable handheld analyzer that contains the calibrator and the reagents in a pre-packaged cartridge.

This analyzer delivers testing for blood gases, electrolytes, coagulation and glucose.

Other common instruments:

Cardiac Markers- is a solid-phase chromatographic assay that allows for qualitative detection of creatine kinase and myoglobin.

Click icon to add pictureUrinalysis reagent strips- are firm plastic strips onto which several separate reagents are affixed.

The test is for the detection of one or more of the following analytes in urine: Ascorbic acid, glucose, bilirubin, ketone, specific gravity, ph, blood, urobilinogen, nitrite and leukocytes.

Blood glucose meter- is an electronic device for measuring the blood glucose level.

HemoCue Albumin Systems- it delivers accurate and fast quantitative determination of low levels of albumin in urine. They are used for screening, diagnosing, monitoring and supporting the clinical evidence in the treatment of microalbuminuria.

Point of Care Analyzer

• used to measure blood gas, pH, electrolytes, and some metabolites in whole blood specimens.

• They are also used to determine abnormal metabolite and/or electrolyte levels in blood and the patient’s acid-base balance and levels of oxygen/carbon dioxide exchange.

• It have extensive test menus and provide a rapid laboratory results to expedite a patient’s diagnosis and treatment.

• There are many compact analyzers available for bedside testing, screening projects, wellness centres, operating rooms and emergency rooms.

Application:• BLOOD GLUCOSE TESTING

• Blood glucose levels are measured by a meter and use a capillary blood directly from finger sticks.

• The blood glucose test is ordered to measure the amount of glucose in the blood right at the time of sample collection. It is used to monitor glucose levels in persons with diabetes.

Drugs of Abuse Testing

• Drug of abuse testing are frequently ordered on patients who exhibit symptoms of intoxication or offer a history of drug ingestion.

• Rapid and accurate results are critical to manage patients effectively.

FACTORS AFFECTING POC ANALYZERS

• Taking the sample from the wrong patient

• Taking the wrong type of sample

• Failure to follow procedure

• Incorrect result interpretation

ADVANTAGES:

• Rapid test results essential for decision-making

• A system that generates a printout of the results

• Requires small sample volume• Allows testing in a variety of locations• Potential to improve patient outcome

or workflow by having results immediately available

• Less traumatic for the patients• Portable devices are used

DISADVANTAGES:

• Potentially different reference ranges

• Costly to operate• Minimal training of personnel to

operate the instruments• Management of POCT is

challenging• Not all methods are appropriate

for diagnosis or monitoring treatment

Technology

Group 8 mass spec automated analyser and poct the complete version