Detailed ppt for studying biochemistry lab tests for Inborn errors of metabolism for Pediatricians and medical geneticists

1. HPLC
TMS
GCMS
CHAKSHU CHAUDHRY
Senior Resident
GENETIC METABOLIC UNIT
PGIMER
9/11/2021

2. INTRODUCTION -
CHROMATOGRAPHY

3. Chromatography-Basic principle
• Chromatography, literal meaning “colour
writing”
• Chromatography is a process whereby a mixture
of solutes may be resolved into its components
• Achieved by exploiting differences in affinity of
the solutes for particles of an insoluble matrix
over which a solution of the components is
passing.
• The insoluble matrix is called the stationary
phase, while the solution which passes through it
is called the mobile phase

4. • The mobile phase is the phase which moves in a
definite direction.
• It may be a liquid (LC ), a gas (GC)
• The mobile phase consists of the sample being
separated/analyzed and the solvent that moves the
sample through the column
• The stationary phase is the substance which is fixed
in place for the chromatography procedure
• Subtle differences in a compound's partition
coefficient result in differential retention on the
stationary phase and thus changing the separation

5. The liquid is called the mobile phase and the particles the stationary phase.
A mixture of the molecules that shall be separated is introduced into the mobile
phase.
If a mixture containing blue and red molecules is to be separated the mixture
containing these two types of molecules is introduced into the mobile phase in front of
the stationary phase.
The mixture of the red and blue molecules is then transported by the mobile
phase through the stationary phase.
The molecules in the mixture that adsorbs the most to the stationary phase, in this
particular case the red molecules, is moving slowest through the particle bed.
The red molecules become separated from the blue

6. Chromatographic techniques
• Techniques by chromatographic bed shape-
column chromatography, paper
chromatography , thin layer chromatography
• Techniques by physical state of mobile phase-
Liquid, Gas chromatography
• Techniques by separation mechanism-ion
exchange, size exclusion chromatography

7. Paper chromatography –
an analytical technique for
separating and identifying
mixtures that are or can be colored,
especially pigments
Thin Layer chromatography
useful for separating organic compounds
Spotting a TLC plate with
sample
Running the
TLC plate in
solvent
Column chromatography

8. Mixture of two solutes, A and B, dissolved in a suitable buffer solution
A column is filled with beads of an insoluble substance expected to bind differentially and
reversibly to A and B
Mixture applied to the top of the column as a narrow layer
The layer of solutes will pass into the column, and the solutes can be washed out by pouring
more buffer into the column.
Molecules of B which bind strongly will be retarded with respect to the molecules of A.
In time, they will separate into bands, and eluted at different times
Collecting constant volumes of the eluate (which are called fractions), the separated solutes will
be distributed in the different fractions
If fractions 4-9 are combined ("pooled"), a solution of pure A will be obtained
If fractions 12-18 are pooled, a solution of pure B will be obtained
Column chromatography

9. HPLC

10. HPLC and traditional column
chromatography
• HPLC - separation of compounds in a mixture more
efficiently and quickly -
• The separation is more effective due to greater
surface area achieved due to very small particle size
of stationary phase
• flow time and run time due is more due to increased
surface area.
• To minimize this obstacle the high pressure is applied
to the flow of mobile phase through the column by
use of pumps

11. HPLC Instrumentation
• The basic HPLC instrumentation comprises of
components like
• ¤ PUMPS
• ¤ Injection system
• ¤ Columns
• ¤ Detectors and
• ¤ Computer for display and documentation of data

12. • Quantitative Analysis
• In order to make a quantitative assessment of the
compound, a sample with a known amount of the
compound of interest is injected and its peak height
or peak area is measured

13. APPLICATIONS
• HPLC is optimum for the separation of chemical and biological
compounds that are non-volatile
• Typical non-volatile compounds are:
• Pharmaceuticals like aspirin, ibuprofen, or acetaminophen
(Tylenol)
• Salts like sodium chloride and potassium phosphate
• Proteins like egg white or blood protein
• Organic chemicals like polymers (e.g. polystyrene, polyethylene)
• Heavy hydrocarbons like asphalt or motor oil
• natural products such as ginseng, herbal medicines, plant
extracts
• Thermally unstable compounds such as trinitrotoluene (TNT),
enzymes

14. BioMedical uses
• The measurement of specific amino acids in body fluids -
plasma/serum, urine or CSF is used for diagnosing many amino-
acid disorders such as
 Phenylketonuria
 Tyrosinemia
 Maple syrup urine disease
 Homocystinuria
• Non-ketotic hyperglycinemia
 Diagnosis will be made when both CSF and plasma levels of
glycine are elevated.
 Isolated elevation of glycine - other non-genetic causes such as
perinatal asphyxia or bloody tap (due to contamination of CSF
with blood).

15. Advantages of HPLC
• Easy and high specificity
• All samples (plasma, urine or CSF) can be used

16. TMS/
MS/MS

17. Tandem mass spectrometry/ mass
spectrometry-mass spectrometry
(MS/MS )
• Two mass spectrometers are situated in tandem.
• Can detect multiple independent metabolites/analytes in a
single test.
• The basic principle of TMS relies on the ionization and
fragmentation of each molecule or metabolite into specific
ions coupled with a robust detection system which is
computerized to provide results
• Measures amino-acids, organic acids and fatty acids in form of
their acyl-carnitine esters.
• Core of expanded newborn screening

18. • Tandem mass spectrometry is used to produce structural
information about a compound by fragmenting specific
sample ions inside the mass spectrometer and identifying the
resulting fragment ions.
• This information of specific compounds based on their
specific and characteristic fragmentation patterns can then be
pieced together to generate structural information regarding
the intact molecule.

19. Tandem mass spectrometer
• MS/MS consists of two or more mass
spectrometer analyzers all in a single
instrument
• Types-
– quadrupole-quadrupole type (also known as Triple
Quadrupole instruments)
– or the hybrid types: including magnetic
sector/quadrupole, and the quadrupole/time-of-
flight (Q-TOF) geometries

20. DBS collection on filter paper
• Warm the heal by rubbing to increase blood circulation
• Fill in baby s details/ attach a bar code
• Not touch the specimen collection area with fingers
• Wash hands, wear gloves
• Clean area of puncture with alcohol swab and allow to dry
• Sterile lancet/ puncture device – not more than 2mm deep
• Gently wipe away first drop with a dry sterile gauze.
• Donot squeeze – may cause hemolysis, contaminate blood sample with
tissue fluid

21. • Allow drop of blood to form that’s
big enough to fill the circle on the card
• Apply only once to each circle
• Fill on side of card only
• Make sure blood fills circle completely
• Let card dry for 3 hours on a flat surface
at room temp of 18 – 25 degrees C
• Do not stack/ use heat / sunlight
• Check all information filled
• Send to lab within 24 hours of collection

24. • AbSciex QTRAP 4500

26. Parent
ion
Daughte
r ion

28. Limitations
• Does not screen for many IEMs - mitochondriopathies,
purine and pyrimidine disorders, neurotransmitters,
congenital disorders of glycosylation (CDG) and very long
chain fatty acids.
• May show normal metabolite levels as they are not always
deranged. In such cases, the metabolic tests should be
repeated during acute sickness.
• Not specific though highly sensitive, and thus remains a
screening test for majority of disorders.
• Use for prenatal diagnosis is not recommended.

30. GAS CHROMATOGRAPHY

31. • Gas chromatography (GC), is a type of
analytical chromatographic technique used for
separating and analysing compounds that can
be vaporised without decomposition
• A mass spectrometer is an instrument that
produces ions and separates them in the gas
phase according to their mass-to-charge ratio
(m/z))

32. • Principal similar to column chromatography
• The mobile phase is a carrier gas, usually an inert gas
• The stationary phase is a microscopic layer of liquid
or polymer on an inert solid support
• The gaseous compounds being analyzed interact with
the walls of the column, which is coated with
different stationary phases.
• This causes each compound to elute at a different
time, known as the retention time of the compound

33. GC vs Column chromatography
Gas
chromatography
Liquid (HPLC)
Stationary phase liquid Solid
Mobile phase Gas Liquid
Temperature
control
Controlled in oven No control

34. Instrumentation-
Carrier Gas
Flow regulators and meters
Sample injection system
Columns & ovens
Detectors

35. Carrier Gas
• Purpose-sweep sample through the column, protect column from oxygen
exposure at temperature, assist with function of the detector
• Requirements:
 It should be inert and available at low cost
 High purity
 Easily available
 Less risk of explosion or fire hazards
• Egs-mobile phase gas
Helium ,argon ,nitrogen , carbon dioxide and hydrogen
• Selection of the best carrier gas – type of detector, sample type , it effects
both the column separation and detector performance
• Helium-M/C carrier gas used , safe, efficient, works with greater number of detectors,somewhat difficult to obtain
• Hydrogen-Most efficient ,best separation, inflammable

36. Packed columns are fabricated from glass, metal (stainless steel, copper,
aluminum), or Teflon tubes
Typically have Lengths------ 2 to 3 m
Inside diameters ------- 2 to 4 mm
.
These tubes are densely packed with a uniform, finely divided packing
material(diatomaceous earth),solid support, that is coated with a thin layer (0.05
m) of the stationary liquid phase
Columns-Stationary phase
Packed Open tubular or capillary
Typical stationary phases are large molecular weight polysiloxane, polyethylene glycol, or polyester
polymers

37. The column is placed in an oven where the
temperature can be controlled very
accurately over a wide range of
temperatures.
Typically, GC oven temperatures range
from room temperature to 300 degree C
If temperature too high, causes co-elution
• poor resolution but faster separation
If temperature is too low, longer elution
times
• adequate resolution, but a separation that
takes very long
GC oven
with column in place
Temperature Program-The “simplest” way
to alter the separation in GC
Pressure programming of the carrier gas is
less common.

38. Process Flow Schematic
Carrier gas
(nitrogen or
helium)
Sample injection
Long Column (30 m)
Detector (flame
ionization
detector or FID)
Hydrogen
Air

39. GC -MS
• Gas chromatograph connected to a mass
spectrometer which acts as the detector

40. Applications-GCMS
• Quantitation of pollutants in drinking and
wastewater (environmental protection)
• Quantitation of drugs and their metabolites in blood
and urine for both pharmacological and forensic
applications
• Identification of unknown organic compounds in
hazardous waste dumps
• Biochemical diagnosis – detection of organic
compounds in urine.

41. Patient 1
• poor feeding and vomiting in the 1st wk of life;
lethargy and coma.
• hypertonicity with bouts of flaccidity
manifested as repetitive movements of the
extremities (boxing and bicycling).
• H/o sibling death with similar history.

42. –raised Leucine, Isoleucine and Valine

43. 2-Hydroxyisovaleric acid
2-Keto-3-methylvaleric acid
2-Ketoisocaproic acid
2-Hydroxy-3-methylvaleric acid
GCMS Chromatogram of a patient with MSUD
IS

44. Type Age of onset Clinical features
Classic Neonatal Poor feeding
Irritability, lethargy
Opisthotonus
Fencing, bicycling
Obtundation, coma
Maple syrup odour
Intermediate Variable Poor feeding
Irritability
Developmental delay
Encephalopathy
Intermittent Variable Episodic cdecompensation that
can be severe
Thiamine
responsive
Variable Similar to intermediate
enzyme system (branched-chain α-ketoacid
dehydrogenase [BCKDH])
thiamine (vitamin B1) pyrophosphate as a coenzyme.

45. Management
• Acute state – hydration , rapid removal of
BCAA
• Hemodialysis
• Cerebral edema – mannitol/ diuretics
• Synthetic formula / diet low in BCAA

46. Patient 2
• 2weeks - vomiting and severe acidosis in the
first 2 wk of life. Lethargy, convulsions, and
coma may.
• Sepsis was ruled out.
• Family history of 3rd degree consanguinity was
present.
• Acute afebrile encephalopathy

47. Initial investigations(case 1 contd)
• pH=7.1
• pCO2=18
• HCO3=7
• Na = 145
• Cl = 103
• Lactate – 1.4
• Anion gap = 35
• Glucose= 89 mg/dl
• Hb- 11.2 gm%,
• TLC- 8900 (12/80/5/3)
• Platelet count- 90,000
• Urine ketones: 3+
• Blood ketones : 5.9
• NH3=240
• HAGMA
• Ketoacidosis
• Elevated NH3
• Neutropenia
• Thrombocytopenia

48. HAGMA
Fatty acid
oxidation defects
Skin inv No skin inv
Multiple carboxylase/
Biotinidase deficieny
Methylmalonic acidemia
Propionic acidemia
Isovaleric academia
ketosis No ketosis
Organic acidemias
Plasma/urine ketones & Lactic acid
• Cytopenia may help
All these can have mild hyperammonemia

49. Axial T2-weighted MRI - swelling with T2
hyperintensity within bilateral globus
pallidi
• Intellectual
disability
• Neurologic
manifestations :
dystonia,
choreoathetosis,
tremor and
paraparesis

50. Further Investigations/Specialized tests
Tests that look for abnormal amounts of normal metabolites
or abnormal metabolites or both
• Organic acid analysis of urine samples by GC/MS
• Acylcarnitine analysis of serum samples by MS/MS
• Amino acid analysis of serum samples by HPLC or TMS

51. Tandom Mass Spectrometry (MS/MS)
Elevated C3-carnitine (propionyl-carnitine) -
PA
MMA
Cbl defects
Vit B12 defiecuency
• Elevated
Acylcarnitines-
• C3- Propionyl
• C5- Isovaleryl
• C5DC- Glutaryl
• C5OH – 5-OH
Isovaleryl
• C5:1- Tiglyl

52. Methylmalonic acid
IS
Organic acids: Methylmalonic, methylcitric, 3-OH-propionic
Amino acids: Homocystiene – 160 μmol/L (ref range < 12 μmol/L)
Scenario 2 A

53. IS
3HBA
3HPA
3HVA
Elevated propionic acid, propionyl glycine,
hydroxypropionate, methylcitrate
Propionylglycine
PA
Scenario 2 B

54. Scenario 2 C
• If however,
• TMS - The main compound in plasma is
isovalerylcarnitine - dried blood on a filter
paper.

55. IS
3-hydroxyisovaleric acid
isovaleric acid and metabolites (isovalerylglycine, 3-
hydroxyisovaleric acid) - urine.

56. Organic acidemias – Management
• Suppress toxic metabolite production - Stop feeding
• Correct fluid imbalance and electrolyte abnormalities -
Hydration
• Promote anabolism -IV glucose @ 8 mg/kg/m & insulin if
needed
• Correct metabolic acidosis- Liberal bicarbonate therapy
• Correct hyperammonemia – suppress catabolism, Dialysis
• Maintain nutritional status
• Empiric cofactor administration –
– Vitamin B12 – 1 mg
– Biotin 5-20 mg
– Carnitine 100-200mg/kg QID oral or IV OD

57. Scenario 2 D
• Laboratory findings acidosis, ketosis, and
moderate hyperammonemia.
• Organic acidemia was suspected.
• TMS- elevated C5:1 and C5 OH, mild increase
in Glycine

58. 2M3HBA
IS
Methyl
acetoacetate
Tiglylglycine
β-Ketothiolase
The urine contains large amounts of 2-
methylacetoacetate and its decarboxylated products
butanone, 2-methyl-3-hydroxybutyrate, and tiglylglycine

59. • Reversible mitochondrial enzyme is involved in
final steps of catabolism of isoleucine and also
in oxidation of fatty acids.
• Oral l-carnitine (50-100 mg/ kg/24 hr)

60. Scenario 2 E
• Acute crisis - metabolic acidosis and ketosis,
Hypoglycemia, hyperammonemia, and elevations of
serum transaminases
• Neuroimaging of the brain – prominent sylvian
fissures.
• TMS – elevated C5DC Acylcarnitine (Glutaryl)
• Other clues – enlarging head size, macrocephaly,
developmental delay, dystonias.
• Subdural hematoma and retinal hemorrhage
following minor falls and head traumas

61. Glutaric acid
IS
High glutaric acid - urine, blood, and CSF.
3-Hydroxyglutaric acid - urine.

62. • Acute crisis management
• Lysine, tryptophan restricted diet
• Carnitine supplementation
• Movement disorder - Baclofen
Treatment

63. Patient 3
• Profound intellectual disability, Cognitive
delay
• Hyperactive with autistic behaviors, including
purposeless hand movements, rhythmic
rocking, and athetosis.
• Lighter in their complexion
• Seborrheic or eczematoid rash
• Musty or mousey - phenylacetic acid

64. GCMS Chromatogram of Phenylketonuria patient
Phenyllacti
c acid
2-Hydroxyphenylacetic acid
IS
PKU
TMS – high Phenylalanine values

65. 1. Recommended - phenylalanine levels -
• Birth – 12 years : 2 - 6 mg/dL
• Older: 2 - 15 mg/dL
2. LNAAs (tyrosine, tryptophan, arginine, leucine, isoleucine,
valine, methionine, histidine, lysine, threonine and
phenylalanine) share the same transporter protein (LNAA type 1,
LAT-1) for transit through the intestinal cell membrane and
blood–brain barrier.
3. Sapropterin dihydrochloride (Kuvan), a synthetic form of BH4,
reduces phenylalanine levels -10 mg/ kg/day, it
Treatment

66. Patient 4
• 6 month boy - failure to thrive, hepatomegaly,
and coagulation diathesis .
• DDs - galactosemia, hereditary fructose
intolerance, neonatal iron storage disease,
giant cell hepatitis, and citrullinemia type II

67. 4HPLA
LA
PA
4HPAA 4HPPA
IS
GCMS Chromatogram of Tyrosinemia
TMS – elevated Tyrosine levels

68. Hyperphenylalaninemia
• Fumarylacetoacetate hydrolase (type 1)
• Tyrosine aminotransferase (type 2)
• 4-hydroxyphenylpyruvate dioxygenase (4-
HPPD) (type 3)
Acquired –
• Severe hepatocellular dysfunction (liver
failure)
• Scurvy (vitamin C is the cofactor for 4-HPPD)
• Hyperthyroidism.

69. TYPE I Type II (RICHNER-HANHART
SYNDROME, OCULOCUTANEOUS
TYROSINEMIA)
TYPE III
Failure to thrive
Hepatomegaly
Coagulation abnormalities
Acute hepatic crisis
Acute peripheral neuropathy
Fanconi-like syndrome
(hyperphosphaturia,
hypophosphatemia, normal
anion gap metabolic acidosis,
and vitamin D–resistant rickets)
Boiled cabbage odour
Palmar and plantar hyperkeratosis,
Herpetiform corneal ulcers
Intellectual disability
Liver and kidney function are
normal
Developmental delay
Seizures, intermittent
Ataxia
Self-destructive
behavior
Liver and renal
normal
Treatment – phenylalanine and tyrosine-restricted diet,
nitisinone

70. Patient 5
• Child - blackening of the urine on standing/
black stained diapers

71. IS
Phosphoric acid Homogentisic
acid
ALKAPTONURIA

72. • Deficiency of homogentisic acid oxidase
(homogentisate 1,2-dioxigenase)
• Ochronosis and arthritis in adulthood
• Diagnosis is confirmed by finding massive
excretion of homogentisic acid on urine
organic acid testing
• Treatment – phenylalanine and tyrosine-
restricted diet , nitisinone

73. Thank you